D-threo-Pentitol, 2,5-anhydro-1,3,4-trideoxy-2-C-(2,4-difluorophenyl)-4-[[4-[4-[4-[[[1-[(1S,2S)-1-ethyl-2-(phenylmethoxy)propyl]hydrazino]carbonyl]amino]phenyl]-1-piperazinyl]phenoxy]methyl]-1-(1H-1,2,4-triazol-1-yl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February - 05 March 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-D

IUPAC Name:

PAZ-D

Details on test material:

- Name of test material (as cited in study report): PAZ-D
- Substance type: White powder
- Physical state: solid
- Purity: 99.3% (w/w)
- Lot/batch No.:124
- Expiration date of the lot/batch: 13 January 2013
- Storage condition of test material: At room temperature in the dark under nitrogen
- Hygroscopic: Yes
- pH: 5-6 at 20°C (more alkaline than that)
- Stability in vehicle: Not stable in very protic / acidic solvents
- Stability in N,N-Dimethylformamide: Yes
- Solubility in vehicle: Likely to be reasonably soluble in protic / acidic solvents and in di-polar protic solvents

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 labeled 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): 16.1 – 22.5ºC
- Humidity (%): 35 - 67%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Temporary deviations from the minimum level of temperature and minimum relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 08 February - 05 March 2012

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25, 50%

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 10, 25 and 50% were 1.3, 1.1 and 1.0 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 10, 25 and 50% were 595, 487 and 450 DPM respectively. The mean DPM/animal value for the vehicle control group was 453 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 both animals at 50% (Days 2-4), which did not hamper scoring of the skin reactions.

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 both animals at 50% (Days 1-3), 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.

Macroscopy of the auricular lymph nodes and surrounding area:

The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Conclusions:

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.3, 1.1 and 1.0 respectively.
Since there was no indication that the test substance elicited an SI ≥ 3 when tested up to 50%, PAZ-D was considered to be a non skin sensitizer