4-[4-[4-[(3R, 5R)[5-(2,4-difluorophenyl) tetrahydro-5-(1H-1,2,4-triazol-1-yl-methyl)-furan-3-yl]methyloxy]phenyl]-1-piperazinyl]phenyl-2-[(1S,2S)(1-ethyl-2-phenylmethoxypropyl)]-2,4-dihydro-3Htriazol-3-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11- 30 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Paz-E

IUPAC Name:

Paz-E

Details on test material:

- Name of test material (as cited in study report): Paz-E
- Substance type:White powder
- Physical state: solid
- Expiration date of the lot/batch: 25 November 2012
- Storage condition of test material:At room temperature protected from light in desiccator
- Hygroscopic: Yes, in desiccator
- Test substance handling: Use amber-coloured glassware or wrap container in tin-foil.
- pH: 5-7 at 20°C

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeld makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 – 21.4ºC
- Humidity (%): 45 - 72%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 11- 30 January 2012

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 25, 50, 70%

No. of animals per dose:

5

Details on study design:

The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (20011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: All animals surviving to the end of the study were sacrificed by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal).

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 25, 50 and 70% were 1.3, 3.2 and 2.8 respectively

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 70% were 543, 1347 and 1179 DPM respectively. The mean DPM/animal value for the vehicle control group was 414 DPM.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White test substance remnants were present on the dorsal surface of the ears of all animals at 50 and 70% throughout the observation period, which did not hamper scoring of the skin reactions. Based on these results, the highest test substance concentration selected for the main study was a 70% concentration.

Other results - main study:

Skin reactions / Irritation:

No irritation of the ears was observed in any of the animals examined.

White test substance remnants were present on the dorsal surface of the ears of all animals at 25% (Day 3 and 4), 50 and 70% (Days 1-5 and/or 6), which did not hamper scoring of the skin reactions.

Systemic toxicity/Body weights:

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Macroscopy of the auricular lymph nodes and surrounding area:

The auricular lymph nodes of the animals at 50 and 70% appeared larger in size as compared to the control groups.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 70% were 1.3, 3.2 and 2.8 respectively.

These results indicate that the test substance could elicit an SI ≥ 3. An EC3 value (the estimated test substance concentration that will give a SI =3) of 47.4% was calculated.

The response of the 70% group did not follow the expected dose-response relationship which more often seen in these kind of studies. The response might be less due to differences in skin penetration (less vehicle present) or viscosity.