1-amino-N-[6-cyano-5-(trifluoromethyl)-3-pyridyl]cyclobutanecarboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-12-16 to 2016-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

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:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Cas Number:

1950587-17-3

Molecular formula:

C12H11F3N4O

IUPAC Name:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: JNJ-63632283-AAA (T003665)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: I15HD3050
- Expiration date of the lot/batch: 2017-08-17 (retest date)
- Appearance: White powder
- Purity: 98.1% (w/w) (UHPLC determination with specification >=95%)
- Purity test date: 2015-10-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the solid test item was applied directly on top of the skin tissue.

OTHER SPECIFICS
- Correction factor: 1
- pH (1% in water, indicative range): 6.8 – 6.7 (determined by WIL Research Europe)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: 20 female (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19.6 - 23.2 grams
- Housing: group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material . Paper and shelters were supplied as cage-enrichment. On day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet t (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days before the start of treatment, under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2015-12-16 to 2016-01-18

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 2, 25 and 50% (w/w)

No. of animals per dose:

5 females per group; 4 groups

Details on study design:

RANGE FINDING TESTS:
- A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
- Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration that could technically be applied.
- The test system, procedures and techniques were identical as those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on days 1 and 3, and on Day 6.
- Animals were sacrificed after the final observation.
- Pre-screen test results: No irritation and no signs of systemic toxicity were observed in any of the animals examined. White staining of the dorsal surface of the ears by test item remnants was noted for all animals at a concentration of 25% (Days2-4) and 50% (Days2-6). The staining did not hamper the scoring of the ears. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test item concentration selected for the main study was a 50% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT - INDUCTION days 1, 2, 3
- Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.
- The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
- The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

EXCISION OF THE NODES - day 6
- Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of ³H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
- After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING FOR RADIOACTIVITY - day 6
- Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - day 7
- Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item preparations (w/w) were prepared within 4 hours prior to each dosing.
- No adjustment was made for specific gravity of the vehicle.
- Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels.

- Criteria used to consider a positive response:
A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).
Classification of results: UN-GHS 2007; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skin reaction
EC3 value ≤ 2%: sub-category 1A
EC3 value ≥ 2%: sub-category 1B

Positive control substance(s):

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

Statistics:

No statistics available

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by WIL Research Europe. In this study, performed in November 2015, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Hexylcinnamaldehyde. The females were approx. 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKBJ8846V (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v). SI values calculated for the test item concentrations 5%, 10% and 25% were 1.0, 1.5 and 4.4 respectively. An EC3 value of 17.8% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
The raw data, study plan and report from this study are kept in the WIL Research Europe archives. The test described above was performed in accordance with WIL Research Europe Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

0%

Results 2

Key result

Parameter:

SI

Value:

0.9

Variability:

± 0.2

Test group / Remarks:

2%

Results 3

Key result

Parameter:

SI

Value:

0.9

Variability:

± 0.2

Test group / Remarks:

25%

Results 4

Key result

Parameter:

SI

Value:

0.7

Variability:

± 0.1

Test group / Remarks:

50%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 746 ± 113
2% w/w group: mean DPM ± SEM: 669 ± 66
25% w/w group: mean DPM ± SEM: 658 ± 90
50% w/w group: mean DPM ± SEM: 502 ± 47
DPM = Disintegrations per minute
SEM = Standard Error of the Mean

EC3 CALCULATION
It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 50%.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: No irritation of the ears was observed in any of the animals examined. White staining of test item remnants on the dorsal surface of the ears did not hamper scoring for erythema. 
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 
- Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 25 and 50% were 669, 658 and 502 DPM, respectively. The mean DPM/animal value for the vehicle control group was 746 DPM. The SI values calculated for the item concentrations 2, 25 and 50% were 0.9, 0.9 and 0.7, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, JNJ-63632283-AAA (T003665) was not considered to be a skin sensitizer. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United States (2015) (including all amendments) and the Regulation (EC) No 1272/2008 (including all amendments).