1-Pyrrolidinecarboxylic acid, 2-[5-[4'-[2-[(2S)-1-[(1,1-dimethylethoxy)carbonyl]-2-pyrrolidinyl]-1H-imidazol-5-yl][1,1'-biphenyl]-4-yl]-1H-pyrazol-3-yl]-, 1,1-dimethylethyl ester, (2S)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 9th, 2007 to August 9th 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Done by OECD and GLP standards

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl (2S)-2-[5-(4'-{2-[(2S)-1-[(tert-butoxy)carbonyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-[1,1'-biphenyl]-4-yl)-1H-imidazol-2-yl]pyrrolidine-1-carboxylate

EC Number:

941-432-8

Cas Number:

1085706-46-2

Molecular formula:

C36 H44 N6 O4

IUPAC Name:

tert-butyl (2S)-2-[5-(4'-{2-[(2S)-1-[(tert-butoxy)carbonyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-[1,1'-biphenyl]-4-yl)-1H-imidazol-2-yl]pyrrolidine-1-carboxylate

Test material form:

solid: crystalline

Details on test material:

Off white crystalline solid stored at room temperature in the dark.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

At the start of the study the mice were in the weight range of 15-23 grams and were 8-12 weeks old. Animals were acclimatised for at least 5 days prior to the study. Free access to mains tap water and food was allowed through out the study. The temperature and relative humidity were set to
achieve limits of 19-25C and 30-70% respectively. The lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of
darkness.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Test material at concentrations of 5%, 10% or 25% w/w.

No. of animals per dose:

3 groups of five animals were treated at each concentration group and a further group of five animals were treated as a control group with
dimethylformamide alone.

Details on study design:

The mice were treated by daily application of 25 μl of the appropriate concentration of the test material to the dorsal surface of each ear for three
consecutive days (Days 1, 2, 3) by using a micropipette whose tip is used to spread the formulation over the dorsal surface of each ear. A further
group of five mice received the vehichle in the same manner. On Day 6 all mice were injected with 250 ul of a phophate buffered saline solution
containing 3H-methyl thymidine (3HTdR ) to the tail vein giving a total of 20 uCi to each mouse. All animals were observed twice daily on Days 1, 2
and 3 and once daily on days 4 ,5, and 6. Any signs of toxicity or ill health were noted. Body weights of each mouse were recorded before dosing and before termination. Five hours after administration of 3HTdR all mice were killed by carbon dioxide asphyxiation and their auricular lymph nodes were excised and a 1 ml of phosphate buffered saline was added to each set of lymph nodes. Preparation of a single cell suspension was completed for the lymph nodes cells for each individual animal following standard procedures. Determination of the 3HTdR incorporation was completed by measuring radioactive disintegration for each animal's lymph node cells by using a beta-scintillation counter.

Positive control substance(s):

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

Statistics:

Analysis of variance (ANOVA) was carried out on the data followed by Dunnett's test for comparisons made beween the control and treatment groups in the event of a significant result from the ANOVA.

Results and discussion

Positive control results:

Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Concentration of test material in the vehicle at 5 % resulted in a stimulation index (SI) of 1.05 which is a negative result.Concentration of test material in the vehicle at 10 % resulted in a stimulation index (SI) of 1.66 which is a negative result.Concentration of test material in the vehicle at 25 % resulted in a stimulation index (SI) of 2.51 which is a negative result. A stimulation index of less than 3 was recorded for the three concentrations of the test material.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Concentration of test material in the vehicle resulted in a mean disintegration of 861.87 dpm. Concentration of test material in the vehicle at 5 % resulted in a mean disintegration of 903.70 dpm which is a negative result. Concentration of test material in the vehicle at 10 % resulted in a mean disintegration of 1426.65 dpm which is a negative result.Concentration of test material in the vehicle at 25 % resulted in a mean disintegration of 2167.13 dpm which is a negative result.The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups.

Any other information on results incl. tables

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. White residual test material was noted, post dose one to three days after treatment on the ears of animals treated with the test material at a concentration of 10% w/w in dimethyl formamide and post dose on Days 1 and 2 and on Days 3 and 4 in animals treated with the test material at a concentration of 25% w/w in dimethyl formamide.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the test. A stimulation index of less than 3 was recorded for the threeconcentrations of the test material.