N-((3R,4R)-1-benzyl-4-alkylpiperidin-3-yl)-2-halogeno-N-alkyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 December 2007 to 08 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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

Details on test material:

Batch No.: E010005747 or 902/690550/A/1/1
Purity: 94.2%

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V.
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 18.1 - 23.4 g
- Housing: single house, Makrolon Type I cage with wire mesh top, with granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

IN-LIFE DATES: From: 2007-12-18 To: 2008-01-08

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

In preliminary study: 2.5, 5, 10, and 25 % (w/v)
In main study: 5%, 10%, 25% (w/v)

No. of animals per dose:

In preliminary study: Two mice per dose.
In main study: Four mice per dose.

Details on study design:

Preliminary irritation study:
To determine the highest non-irritanting test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 25 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application.

Main study:
1) Allocation
Three groups of four animals were treated with one test substance concentration per group (10, 25 and 50% w/v). One group of four animals was treated with vehicle.
2) Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in dimethylformamide. The application volume, 25 μL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
3) Administration of 3H-Methyl Thymidine
Five days after the first topical application, all mice were administered with 250 μL of 80.4 μCi/mL 3HTdR by intravenous injection via a tail vein.
4) Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

None stated

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The SI value for the experimental group treated with test substance concentrations 5% was 1.61. The SI value for the experimental group treated with test substance concentrations 10% was 1.57. The SI value for the experimental group treated with test substance concentrations 25% was 2.77.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The mean DPM/animal value for the vehicle control group was 746.3 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 5% was 1202.3 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 10% was 1171.8 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 25% was 2063.5 DPM.

Any other information on results incl. tables

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

All treated animals survived the scheduled study period and no signs of toxicity were observed.

Calculation and Results of Individual Data

Test item concentration % (w/v)	Group	DPM per animal	S.I.
	1	746.3
5	2	1202.3	1.61
10	3	1171.8	1.57
25	4	2063.5	2.77

1 = Control Group, 2-4 = Test Group, S.I. = Stimulation Index

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance did not elicit SI ≥ 3, was found to be not a skin sensitiser under the described conditions.