5-Thiazolecarboxamide, N-(2-chloro-6-methylphenyl)-2-[(6-chloro-2-methyl-4-pyrimidinyl)amino]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2003 to 30 October 2003

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:

2003

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 April 2002

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

Performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February, 2000 [RS 813.016.5] based on the OECD Principles of GLP, as revised in 1997 and adopted by decision of the OECD Council [C(97) 186/Final].

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide

EC Number:

695-686-7

Cas Number:

302964-08-5

Molecular formula:

C16 H13 C12 N5 O S

IUPAC Name:

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide

Test material form:

other: solid

Details on test material:

Beige solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CB/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

25 healthy female CBA/CaOlaHsd mice in the weight range 16 g - 24 g and approximately 8 - 12 weeks at beginning of acclimatization were obtained. Acclimatization; Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

UPDATE all of the following- fill in what you can.

Room no. E21 / RCC ltingen

Conditions: Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 ±. 3 °C, relative
humidity 30 - 70 % and 10 - 15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light I 12 hour dark cycle with at least 8 hours music during the light period.

Accommodation: In groups of five in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).

Diet: Pelleted standard Kliba 3433, batch no. 40/03 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC.

Water: Community tap water from ltingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1 %, 5 %, 10 % and 25 % (w/v).

25 % (w/v) was the highest technically achievable concentration in the chosen vehicle.

No. of animals per dose:

Groups of five mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3).

A positive control group of mice was treated with a same volume of 25 % (w/v) ALPHA­ HEXYLCINNAMALDEHYDE in N,N-dimethylformamide (DMF)
A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Details on study design:

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 N,N-dimethylformamide (DMF). The application volume, 25 µI, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A positive control group of mice was treated with a same volume of 25 % (w/v) ALPHA­ HEXYLCINNAMALDEHYDE in N,N-dimethylformamide (DMF). A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µI of
79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Composition: Pentobarbita!um natricum 162 mg, Conserv. (Alcohol benzylicus}, Excip. ad solut. lniect. pro 1 ml) (Veterinaria AG, Zurich).
The draining lymph nodes were rapidly excised and pooled for each individual animal (2 nodes per mouse). Single cell suspensions (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 glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The scintillation counter expresses 3HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).

Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Composition: Pentobarbita!um natricum 162 mg, Conserv. (Alcohol benzylicus}, Excip. ad solut. lniect. pro 1 ml) (Veterinaria AG, Zurich).
The draining lymph nodes were rapidly excised and pooled for each individual animal (2 nodes per mouse). Single cell suspensions (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 glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).

In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality I Viability; Twice daily from acclimatization start to the termination of in-life phase
Body weights; Prior to the 1'' application and prior to necropsy
Clinical signs (local I systemic); Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.

Positive control substance(s):

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

Statistics:

T-Test and Dunnett-test were conducted for assessment of the difference signficance between the test item group and the positive control grop and the negative control group. Mean and standard deviations of the body weight and the DPM values were determined..

Results and discussion

Positive control results:

α-Hexylcinnamaldehyde, was considered to be a sensitiser under the conditions of the test with test control ration obtained for 25% v/v HCA at 9.2.
The test substance is regarded as a sensitizer if at least one concentration of the chemical results in a three-fold greater increase in 3HTdR incorporation compared to control values.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Group I %(w/v) I DPM/mouse M ± SD I S.I. (SD) I Statistical Analysis a) t-test (G = 2, N = 10, t = 2.31) bl Dunnett-test (G = 4, N = 20, t = 2.59) t value Conclusion NCG 1 - 669 ± 163 - - -- PCG 2 25 (HCA) 6179 ± 1816 9.2 (2.7) 6.76 a) • TG 3 5 1173 ± 277 1.8 (04) 2.35 b) -- TG 4 10 1426 ± 465 2.1 (0.7) 3.53 b) •• TG 5 25 1682 ± 376 2.5 (0.6) 4.72 b) •• ** significant difference at p s 0.05 (two sides) -- no significant difference at p s 0.05 (two sides) NCG = Negative control group; PCG = Positive control group; TG = Test group DPM = 3HTdR incorporation as the number of radioactive disintegrations per minute The stimulation index for the positive control substance hexyl cinnamic aldehyde (HCA), was 9.2 which demonstrated the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Group I %(w/v) I DPM/mouse M ± SD I S.I. (SD) I Statistical Analysis a) t-test (G = 2, N = 10, t = 2.31) bl Dunnett-test (G = 4, N = 20, t = 2.59) t value Conclusion NCG 1 - 669 ± 163 - - -- PCG 2 25 (HCA) 6179 ± 1816 9.2 (2.7) 6.76 a) • TG 3 5 1173 ± 277 1.8 (04) 2.35 b) -- TG 4 10 1426 ± 465 2.1 (0.7) 3.53 b) •• TG 5 25 1682 ± 376 2.5 (0.6) 4.72 b) •• ** significant difference at p s 0.05 (two sides) -- no significant difference at p s 0.05 (two sides) NCG = Negative control group; PCG = Positive control group; TG = Test group DPM = 3HTdR incorporation as the number of radioactive disintegrations per minute

Any other information on results incl. tables

There were no deaths and no signs of ill health or toxicity observed during this study.

However the following observations were noted:

No signs of irritation were seen over the dosed area during the study.

Wet fur around the cranial region was noted for all negative control and test animals dosed at 10 and 25% w/v and 3 animals dosed at 50% w/v post-dose from Day 1 or 2. White dose residue on the ears was seen post dose from Day 1 in all animals dosed at 25 or 50% w/v.

Greasy fur around the cranial region was recorded for all animals from the positive control group post dose from Day 1. These signs had resolved completely in all animals by Day 6.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item BMS 540268 was found to be a non-sensitizer when tested at up to the highest achievable concentration of 25 % (w/v) in N,N-dimethylformamide (DMF).
Positive control item ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at concentration of 25 % (w/v) in N,N-dimethylformami de (DMFl.
The test item BMS 540268 causes slight to moderate skin irritation at concentrations of 10 %
and 25 % (w/v) on the ear dorsum of mice.

Executive summary:

In this study STIMULATION INDICES of 1.8, 2.1, and 2.5 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in N,N-dimethylformamide (DMF).

STIMULATION INDEX of 9.2 was determined with the positive control item ALPHA­ HEXYLCINNAMALDEHYDE at concentration of 25 % (w/v) in N,N-dimethylformamide (DMF).

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

The test item BMS 540268 was found to be a non-sensitizer when tested at up to the highest achievable concentration of 25 % (w/v) in N,N-dimethylformamide (DMF).

Positive control item ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at concentration of 25 % (w/v) in N,N-dimethylformami de (DMFl.

The test item BMS 540268 causes slight to moderate skin irritation at concentrations of 10 %

and 25 % (w/v) on the ear dorsum of mice.