Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Feb 2014 to 11 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

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:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

Rj (SPF)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Sex: Females
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old (age-matched, within one week)
- Weight at study initiation: 19.0-19.9 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
- Housing: Group caging / mice were provided with glass tunnel-tubes
- Diet: autoclavable Complete diet for rats/mice – Breeding and maintenance. Ad libitum
- Water: tap water. Ad libitum
- Acclimatisation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 25.3
- Humidity (%): 28 - 70
- Air changes (per hr): 15-20
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

- IN-LIFE DATES: From: 05 Feb 2014 To: 11 Feb 2014

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

50, 25 and 10 % w/v

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
A Preliminary Irritation/ Toxicity Test was performed on CBA/J Rj mice using two doses, at test material concentrations of 100 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 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on day 1 (pre-dose), day 3 (approximately 48 hours 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.

MAIN STUDY
Substance Administration:
- Each mouse was topically dosed on the dorsal surface of each ear with 25 μL of the test material applied using a pipette. Each animal was dosed once a day for 3 consecutive days (days 1, 2 and 3). There was no treatment on days 4, 5 and 6.

Proliferation Assay:
- Thymidine (3HTdR) Administration: on day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile phosphate buffered saline (PBS) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and preparation of draining auricular lymph nodes: 5 hours (± 30 minutes) after intravenous injection, the mice were euthanised. The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of single cell suspension of lymph node cells: a single cell suspension of lymph node cells (LNCs) was prepared and collected in disposable tubes 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 (up to 10 mL). Pooled LNCs were pelleted by centrifuging. Pellets were resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice.
- Determination of incorporated 3HTdR: pellets were gently agitated, re-suspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight incubation at 2-8 °C, precipitates were centrifuged and supernatants were removed. Pellets were re-suspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured.

Observations:
- Clinical observations: all animals were observed at least once daily for any clinical signs, including local irritation and systemic toxicity.
- Body weight: Individual body weights were recorded on day 1 (beginning of the assay) and day 6 (prior to 3HTdR injection) with a precision of +/- 0.1 g.
- Ear thickness: measured by ear punch weight determinations, which was performed on day 6 after the animals were humanely killed.

Terminal Procedures:
- Five hours after intravenous injection, the mice were euthanised by asphyxiation by a rising concentration of carbon dioxide, followed by confirmation of death.

Positive control substance(s):

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

Statistics:

Disintegrations per minute (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

10 % test material

Results 2

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

25 % test material

Results 3

Key result

Parameter:

SI

Value:

8.5

Test group / Remarks:

50 % test material

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. Rigid ears were observed in the 50 % (w/v) dose group on Days 2-5 and in the 25 % (w/v) group on Days 2-3. Slight alopecia was observed in the 50 % (w/v) dose group on Days 4-6. Test material precipitate was detected on the ears of the animals in the 50 % (w/v) dose group on Days 1-6, in the 25 % (w/v) group on Days 1-4 and in the 10 % (w/v) group on Days 2-3.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on body weight, however marked body weight loss (>5 %) was detected for one animal in the 10 % (w/v) dose group.
 
EAR THICKNESS MEASUREMENT
The ear punch weights were out of the historical control range for two animals in the 50 % (w/v) dose group, however, they did not exceed the limit of positive response (upper limit of historical control range + 25%).
 
PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 10 % (w/v) dose group. Larger than normal lymph nodes were observed for 1 animal in the 50 % (w/v) dose group. Slightly enlarged lymph nodes were detected for three animals in the 50 % (w/v) dose group and for all animals in 25 % (w/v) dose group.The stimulation index values were 8.5, 2.6 and 1.5 at concentrations of 50, 25 and 10 % (w/v), respectively.
 
INTERPRETATION OF OBSERVATIONS
The test item was a solid, which was formulated in DMSO. Since, there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation index observed above the threshold limit of 3 at the concentration of 50 % (w/v) (SI value is 8.5) under these exaggerated test conditions were considered to be good evidence that the test material is a sensitiser. No EC3 value was derived, however it can be calculated from the results to be >2%.

Any other information on results incl. tables

Preliminary Screening Test

No mortality or signs of systemic toxicity were detected. Rigid ears were observed in both dose groups on Day 3 and alopecia was detected for both animals in the 100 % (w/v) group on Days 5-6. Alopecia on the crown was observed for one animal in the 50 % (w/v) group on Days 2-6. No marked body weight loss was detected for the animals. Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6. Increased ear thickness values (>25 % increase) were detected in the 100 % (w/v) dose group indicating excessive local skin irritation. The appearance of the lymph nodes was normal in both dose groups. Based on these results, 100 % (w/v) dose was considered too high due to the increased ear thickness values indicating excessive local skin irritation. Therefore, 50, 25 and 10 % (w/v) doses were examined in the main test.

Table 1. Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test).

Animal Number	Test Group	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# %
1	100 % (w/v)	23.5	24.6	4.7
2	100 % (w/v)	23.9	24.3	1.7
	Mean	23.7	24.5	3.2
3	50 % (w/v)	23.6	24.1	2.1
4	50 % (w/v)	23.1	23.1	0.0
	Mean	23.4	23.6	1.1

Notes:

1. *: Terminal body weights were measured on Day 6.

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Table 2. Individual ear thickness for all animals (Preliminary Irritation/Toxicity Test).

Animal Number	Test Group	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
		Right	Left	Right	Left	Right	Left
1	100 % (w/v)	0.20	0.21	0.37	0.35	0.25	0.23	19.77
2	100 % (w/v)	0.21	0.22	0.38	0.32	0.24	0.21	21.73
3	50 % (w/v)	0.22	0.21	0.23	0.23	0.24	0.23	23.21
4	50 % (w/v)	0.21	0.22	0.23	0.24	0.23	0.23	22.69

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).

Table 3. Summarised Clinical Observations (Preliminary Irritation/Toxicity Test).

Period	Animal Number	Test Group	Clinical observations before treatment	Clinical observations after treatment
Day 1	1	100 % (w/v)	Symptom free, ES: 0	Symptom free*, ES i.s.: 0
	2	100 % (w/v)	Symptom free, ES: 0	Symptom free*, ES i.s.: 0
	3	50 % (w/v)	Symptom free, ES: 0	Symptom free, ES: 0
	4	50 % (w/v)	Symptom free, ES: 0	Symptom free, ES: 0
Day 2	1	100 % (w/v)	Symptom free*, ES: 0	Symptom free*, ES i.s.: 0
	2	100 % (w/v)	Symptom free*, ES: 0	Symptom free*, ES i.s.: 0
	3	50 % (w/v)	Alopecia on the crown**, ES: 0	Alopecia on the crown*, ES: 0
	4	50 % (w/v)	Symptom free**, ES: 0	Symptom free*, ES: 0
Day 3	1	100 % (w/v)	Symptom free*, ES: 0	Rigid ears*, ES: i. s.: 0
	2	100 % (w/v)	Symptom free*, ES: 0	Rigid ears*, ES: i. s.: 0
	3	50 % (w/v)	Alopecia on the crown*, ES: 0	Alopecia on the crown, rigid ears*, ES: 0
	4	50 % (w/v)	Symptom free*, ES: 0	Rigid ears*, ES: 0
Day 4	1	100 % (w/v)	Symptom free*, ES: 0
	2	100 % (w/v)	Symptom free*, ES: 0
	3	50 % (w/v)	Alopecia on the crown*, ES: 0
	4	50 % (w/v)	Symptom free*, ES: 0
Day 5	1	100 % (w/v)	Slight alopecia*, ES: 0
	2	100 % (w/v)	Slight alopecia*, ES: 0
	3	50 % (w/v)	Alopecia on the crown**, ES: 0
	4	50 % (w/v)	Symptom free**, ES: 0
Day 6	1	100 % (w/v)	Alopecia**, ES: 0
	2	100 % (w/v)	Alopecia**, ES: 0
	3	50 % (w/v)	Alopecia on the crown, ES: 0
	4	50 % (w/v)	Symptom-free, ES: 0

*: test item precipitate; **: minimal amount of test item precipitate, i. s.: Score made on inner surface of ear

Main Test Estimation of the Proliferation Response of Lymph Node Cells

Table 1. The radioactive disintegrations per minute per animal and the stimulation index. The stimulation index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

Test Group Name	Measured DPM / group	DPM	Number of Lymph Nodes	DPN	Stimulation Index
Background/ (5 % (w/v) TCA)	34/ 36	-	-	-	-
Negative control DMSO	1912	1877.0	8	234.6	1.0
Isopyrazam 50 % (w/v) in DMSO	15923	15888.0	8	1986.0	8.5
Isopyrazam 25 % (w/v) in DMSO	4832	4797.0	8	599.6	2.6
Isopyrazam 10 % (w/v) in DMSO	2764	2729.0	8	341.1	1.5

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test material tested in a suitable vehicle, was shown to have skin sensitisation potential (sensitiser) in the Local Lymph Node Assay. No EC3 value was derived, however it can be calculated from the results to be >2%.

Executive summary:

The skin sensitisation potential of the test material following dermal exposure to female CBA/J Rj mice was determined using the LLNA method according to OECD TG 429 following GLP principles . The test material solutions (50, 25, 10% test material formulated in DMSO) were applied to 4 females per group on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (days 1, 2 and 3). The negative control group received the vehicle (DMSO). There was no treatment on days 4, 5 and 6. On day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. Rigid ears were observed in the 50 % dose group on days 2-5 and in the 25 % group on days 2-3. Slight alopecia was observed in the 50 % dose group on days 4-6. Test material precipitate was detected on the ears of the animals in the 50 % dose group on days 1-6, in the 25 % group on days 1-4 and in the 10 % group on days 2-3. The appearance of the lymph nodes was normal in the negative control group and in the 10 % dose group. Larger than normal lymph nodes were observed for 1 animal in the 50 % dose group. Slightly enlarged lymph nodes were detected for 3 animals in the 50 % dose group and for all animals in 25 % dose group. The observed stimulation index values were 8.5, 2.6 and 1.5 at concentrations of 50, 25 and 10 %, respectively.

The test material tested in a suitable vehicle, was shown to have skin sensitisation potential (sensitiser) in the Local Lymph Node Assay. No EC3 value was derived, however it can be calculated from the results to be >2%.