1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Endpoint summary

Description of key information

Skin sensitisation (OECD 429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 - 28 Mar 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI National Institute for Quality- and Organizational Development in Healthcare and Medicines, Budapest, Hungary

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier, Genest-St-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (main study), 11 weeks (preliminary experiment)
- Weight at study initiation: 20.3 - 21.7 g (main study), 20.5 - 22.8 g (preliminary experiment)
- Housing: groups in type II polypropylene/ polycarbonate cages, Lignocel® 3/4-FASERN Hygienic Animal Bedding (J. Rettenmaier & Söhne GmbH & Co.KG, Rosenberg, Germany), glass tunnel-tubes
- Diet: ssniff® SM Rat/Mouse (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water from the municipal supply, ad libitum
- Acclimation period: 20 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 24.8
- Humidity (%): 30 - 73
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light):12/12

Vehicle:

dimethyl sulphoxide

Concentration:

25 and 50% (w/v) (preliminary irritation/toxicity test)
10, 25 and 50% (w/v) (main test)

No. of animals per dose:

2 (preliminary irritation/toxicity test)
4 (main test)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short preliminary compatibility test. Due to the physical characteristics of the test item, 100% concentration was not achievable. No proper formulation was achieved at 50% (w/v) concentration using acetone: olive oil 4:1 (v/v) mixture, N,N-dimethylformamide, methyl ethyl ketone, propylene glycol or 1% aqueous pluronic PE9200 as vehicle. However, the formulation at 50% (w/v) using dimethyl sulfoxide as vehicle was suitable for the test.
- Irritation: A preliminary irritation/toxicity test was conducted in 2 animals per dose at concentrations of 25 and 50% (w/v) in DMSO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and no radioactive proliferation assay was performed. All mice were observed daily for local irritation at the application site. Alopecia was observed in the 50% (w/v) dose group on Day 6. Test item precipitate was observed on the ears for the both animals in the 50% (w/v) group on Days 1-6 and in the 25% (w/v) dose group on Days 1-4. Rigid ears was observed in the 50% (w/v) dose group on Days 2-5, and in the 25% (w/v) dose group on Days 2-3 which may have been caused by test item remaining on the ears. No irritation was observed.
- Systemic toxicity: All mice were observed daily for any clinical signs of systemic toxicity. No mortality or clinical signs of systemic toxicity were observed.
- Ear thickness measurements: Ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 (before treatment, approximately 48 h after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weights were over the historical control range which may have been caused by test item remaining on the ears.
- Erythema scores: No erythema was observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: tritiated thymidine (³HTdR) incorporation determined by beta-scintillation
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled: Exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index and the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: On Day 1, 25 µL of the test substance freshly diluted with DMSO was topically applied to the dorsal surface of each ear of each mouse. The application was repeated on Days 2 and 3. There was no treatment on Days 4, 5 and 6. On day 6 an intravenous injection of 250 µL phosphate buffered saline (PBS) containing approximately 20 µCi of ³HTdR was made into the tail vein of each experimental mouse. 5 h (± 30 min) later, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of pooled lymph node cells was prepared from each mouse and collected by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS. Pooled LNCs were washed twice via pelleting using centrifugation and resuspension in PBS. Pellets were resuspended in 5% trichloroacetic acid and precipitation was conducted overnight at 2 - 8°C. Following centrifugation pellets were resuspended in 5% trichloroacetic acid and dispersed by using an ultrasonic bath.

Positive control substance(s):

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

Positive control results:

The positive control (25% (w/v) alpha-hexylcinnamaldehyde in DMSO) induced a significant lymphoproliferative response with a stimulation index value of 13.2. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Thus, the results of the positive control group demonstrated the appropriate performance of the assay.

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10% (w/v)

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

25% (w/v)

Key result

Parameter:

SI

Value:

5.6

Test group / Remarks:

50% (w/v)

Key result

Parameter:

EC3

Value:

21.7

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Appearance of the lymph nodes was normal in the 25 and 10% (w/v) dose groups and in the negative control group. Larger than normal lymph nodes were observed in the 50% (w/v) dose group and in the positive control group. The number of radioactive disintegrations per minute (DPM) were measured and corrected with the background DPM value consisting of 5% (w/v) TCA solution measurements. The results were expressed as DPN (DPM divided by the number of lymph nodes) and are shown in table 1.

DETAILS ON STIMULATION INDEX CALCULATION: The stimulation index was calculated by division of the DPN value of a treated group with the DPN value of the negative control group. The SI values for the respective treatment groups are shown in table 1.

EC3 CALCULATION: The calculation of the EC3 value was conducted by linear interpolation according to the equation: EC3 = c + [(3-d)/(b-d)] x (a-c), where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot have the co-ordinates (a,b) and (c,d) respectively. The calculated EC3 value for the test substance is 21.7% (w/v).

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. Alopecia was observed in the 50% (w/v) dose group on Days 2 - 6 and in the 25% (w/v) dose group on Days 3 - 6. Test item precipitate was observed on the ears for the animals in all treated group on Days 1 - 6. Rigid ears were observed in the 50% (w/v) dose group on Days 2 - 6.

BODY WEIGHTS: No treatment-related effects were observed on animal body weights.

Table 1: Results of the LLNA

Test group	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	34	-	-	-	-
Vehicle control (DMSO)	1610	1576.0	8	197.0	1.0
Test substance 50% (w/v)	8925	8891.0	8	111.4	5.6
Test substance 25% (w/v)	5427	5393.0	8	674.1	3.4
Test substance 10% (w/v)	2522	2488.0	8	311.0	1.6
Positive control (25% HCA in DMSO)	20873	20839.0	8	2604.9	13.2

TCA = trichloroacetic acid, HCA = alpha-Hexylcinnamaldehyde 

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

CLP: Skin sens 1B, H317

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jun - 14 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 22 July 2010

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

amended 6 July 2012

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone, Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (main study), 13 weeks (preliminary experiment I), 11 weeks (preliminary experiment II)
- Weight at study initiation: 19.0 - 20.7 g (main study), 21.4 - 23.0 g (preliminary experiment I), 23.2 - 23.7 g (preliminary experiment II)
- Housing: individual type II polypropylene/ polycarbonate cages, Lignocel® 3/4-FASERN Hygienic Animal Bedding (J. Rettenmaier & Söhne GmbH & Co.KG, Rosenberg, Germany), glass tunnel-tubes
- Diet: ssniff® SM Rat/Mouse (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water from the municipal supply, ad libitum
- Acclimation period: 21 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.4 - 26.2
- Humidity (%): 26 - 84
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light):12/12

Vehicle:

dimethyl sulphoxide

Concentration:

50 and 25% (w/v) (preliminary irritation/toxicity test I)
10 and 5% (w/v) (preliminary irritation/toxicity test II)
10, 25 and 50% (w/v) (main test)

No. of animals per dose:

2 (preliminary irritation/toxicity test I and II)
7 (main test)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short preliminary compatibility test. Due to the physical characteristics of the test item, 100% concentration was not achievable. No proper formulation was achieved at 50% (w/v) concentration using acetone: olive oil 4:1 (v/v) mixture, N,N-dimethylformamide, methyl ethyl ketone, propylene glycol or 1% aqueous pluronic PE9200 as vehicle. However, the formulation at 50% (w/v) using dimethyl sulfoxide as vehicle was suitable for the test.
- Irritation: Preliminary irritation/toxicity tests were conducted in 2 animals per dose at concentrations of 25 and 50% (w/v) in DMSO in the preliminary irritation/toxicity test I, and 10 and 5% in DMSO in the preliminary irritation/toxicity test II. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and no radioactive proliferation assay was performed. All mice were observed daily for local irritation at the application site. Alopecia was observed in the 50% (w/v) dose group on Days 3-6. No irritation was observed. Rigid ears were observed for both animals of the 50% (w/v) and for both animals of the 10% (w/v) dose groups on Day 3, slightly rigid ear was observed for both animals of the 25% (w/v) and for both animals of the 5% (w/v) dose groups on Day 3. Test item precipitate or minimal amount of test item precipitate was observed for both animals of the 50% (w/v) dose group on Days 1-6; for both animals of the 25% (w/v) dose group on Days 1-3 or Days 1-5; for both animals of the 10% (w/v) on Days 1-5, and for both animals of the 5% (w/v) dose group on Days 1-4.
- Systemic toxicity: All mice were observed daily for any clinical signs of systemic toxicity. No mortality or clinical signs of systemic toxicity were observed.
- Ear thickness measurements: Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The detected ear thickness values clearly indicated excessive local irritation (≥ 25 %) for both animals of the 50% (w/v) dose group on Day 6, however test item precipitate was present on the ears of the animals during the experiment (Days 1-6) which may have interfered with the measurement; the detected ear thickness values indicated excessive local irritation (≥ 25 %) for both animals (except for one animal's left ear) of the 25% (w/v) dose group on Day 6. The
ear thickness values of other dose groups were within the acceptable range (below the excessive local irritation (≥25%)).
The revealing ear punch weights were within the acceptable range (below the excessive local irritation (≥25%)) in all examined dose groups.
- Erythema scores: No erythema was observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: tritiated thymidine (³HTdR) incorporation determined by beta-scintillation
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled: Exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index and the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: On Day 1, 25 µL of the test substance freshly diluted with DMSO was topically applied to the dorsal surface of each ear of each mouse. The application was repeated on Days 2 and 3. There was no treatment on Days 4, 5 and 6. On day 6 an intravenous injection of 250 µL phosphate buffered saline (PBS) containing approximately 20 µCi of ³HTdR was made into the tail vein of each experimental mouse (except of 2 animals/ dose group). 5 h (± 30 min) later, the mice were euthanized by asphyxiation with ascending doses of carbon dioxide. Once deep anaesthesia was confirmed, the draining auricular lymph nodes were excised. A single cell suspension of lymph node cells was prepared from each mouse and collected by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS. LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4°C. After centrifugation, supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal. Pellets were resuspended in 5% trichloroacetic acid and precipitation was conducted overnight at 2 - 8°C. Following centrifugation pellets were resuspended in 5% trichloroacetic acid and dispersed by using an ultrasonic bath.

Positive control substance(s):

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

Positive control results:

The positive control (25% (w/v) alpha-hexylcinnamaldehyde in DMSO) induced a significant lymphoproliferative response with a stimulation index value of 6.7. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Thus, the results of the positive control group demonstrated the appropriate performance of the assay.

Key result

Parameter:

SI

Value:

7.1

Test group / Remarks:

50 % (w/v)

Key result

Parameter:

SI

Value:

5.8

Test group / Remarks:

25 % (w/v)

Key result

Parameter:

SI

Value:

3.5

Test group / Remarks:

10 % (w/v)

Key result

Parameter:

EC3

Value:

8.2

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The appearance of the lymph nodes was normal in the negative control group and in the 10% (w/v) dose group. Larger than normal lymph nodes were observed in the positive control group and in the 50% (w/v) and 25% (w/v) dose groups.
The number of radioactive disintegrations per minute (DPM) were measured and corrected with the background DPM value consisting of 5% (w/v) TCA solution measurements. The results were expressed as DPN (DPM divided by the number of lymph nodes) and are shown in table 1.

DETAILS ON STIMULATION INDEX CALCULATION: The stimulation index was calculated by division of the DPN value of a treated group with the DPN value of the negative control group. The SI values for the respective treatment groups are shown in table 1.

EC3 CALCULATION: The calculation of the EC3 value was conducted by Log-linear extrapolation according to the equation: EC3ex = 2^{log2(c) + [(3-d)/(b-d)] x [log2(a) - log2(c)]}, where the lowest data point (lying immediately above the SI value of 3 on the LLNA dose-response plot) has the co-ordinates (c,d), and the next higher data point has the co-ordinates (a,b). The calculated EC3 value for the test substance is 8.2 % (w/v).

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity was observed during the main test. Test item precipitate or minimal amount of test item precipitate was observed for all animals of the 50% (w/v) dose group and for five animals of the 25% (w/v) dose groups on Days 1-6 and for two animals of the 25% (w/v) on Days 1-5 and for all animals of the 10% (w/v) dose groups on Days 1-5. Alopecia around the ears was observed for one animals of the 50% (w/v) dose group on Days 3-6 and for five animals of the 50% (w/v) dose group on Days 5-6 and alopecia around the ears was observed for one animal of the 50% (w/v) dose group on Day 5 and extensive alopecia was observed for this animal on Day 6. Alopecia around the ears was observed for one animal of the 25% (w/v) dose group on Days 4-6. The ear thickness data showed a mean increase of >25% at the 50% (w/v) and 25% (w/v) dose levels (indicating potential irritation).

BODY WEIGHTS: No treatment-related effects were observed on animal body weights.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

CLP: Skin sens 1B, H317

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Reliable studies regarding skin sensitisation are available for the test substance. A study for respiratory sensitisation potential is not available.

In vivo:

- LLNA:

The skin sensitisation potential of the test substance was assessed in a mouse local lymph node assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP (M-462501-01-1). The test substance was formulated in DMSO with 50% (w/v) as the highest achievable concentration. The solution was applied on the dorsal surface of both ears of three groups with 4 female CBA/J Rj mice each (25 µL/ear) for three consecutive days (Days 1, 2 and 3) at concentrations of 50, 25 and 10% (w/v). A vehicle control group of 4 females received DMSO. The positive control group (4 females) received 25% (w/v) α-Hexylcinnamaldehyde (HCA) in DMSO. There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality, systemic toxicity or treatment-related effects on body weight were observed in any treated group during the study. There were no indications of any irritancy at the site of application. Alopecia was observed in the 50% (w/v) dose group on Days 2-6 and in the 25% (w/v) dose group on Days 3-6. Test item precipitate was observed on the ears for the animals in all treated groups on Days 1-6. Rigid ears were observed in the 50% (w/v) dose group on Days 2-6. The observed SI values were 5.6, 3.4 and 1.6 at concentrations of 50, 25 and 10% (w/v), respectively. Based on these data, the calculated EC3 value is 21.7%. A significant lymphoproliferative response (SI value of 13.2) was noted for the positive control confirming the validity of the assay. Thus, under the conditions of the study, the test substance was shown to have sensitisation potential and a classification as Skin Sensitizer Category 1B according to Regulation (EC) No 1272/2008 is triggered.

In a second LLNA assay (according to OECD Guideline 429 and in compliance with GLP) the skin sensitisation potential of the test substance was assessed (M-570902 -01 -1).

The test substance was formulated in DMSO with 50% (w/v) as the highest achievable concentration. The solution was applied on the dorsal surface of both ears of three groups with 7 female CBA/J Rj mice each (25 µL/ear) for three consecutive days (Days 1, 2 and 3) at concentrations of 50, 25 and 10% (w/v). A vehicle control group of 5 females received DMSO. The positive control group (5 females) received 25% (w/v) α-Hexylcinnamaldehyde (HCA) in DMSO. There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality, systemic toxicity or treatment-related effects on body weight were observed in any treated group during the study. Alopecia was observed in the 50% (w/v) dose group on Days 3-6 and in the 25% (w/v) dose group on Days 4-6. The ear thickness data showed a mean increase of >25% at the 50% (w/v) and 25% (w/v) dose levels (indicating potential dose level of irritation). The 10% (w/v) was a non-irritating dose.

Test item precipitate was observed on the ears for the animals in all treated groups on Days 1-6. The observed SI values were 7.1, 5.8 and 3.5 at concentrations of 50, 25 and 10% (w/v), respectively. Based on these data, the calculated EC3 value is 8.2%. A significant lymphoproliferative response (SI value of 6.7) was noted for the positive control confirming the validity of the assay. Thus, under the conditions of the study, the test substance was shown to have sensitisation potential and a classification as Skin Sensitizer Category 1B according to Regulation (EC) No 1272/2008 is triggered.

In consideration of the results of the LLNAs the test substance is classified as Skin Sensitizer Category 1B according to Regulation (EC) No 1272/2008.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance is classified as Skin Sensitiser Category 1B according to Regulation (EC) 1272/2008.