(+-)-2,4-dihydro-4-[4-[4-(4-methoxyphenyl)-1-piperazinyl]phenyl]-2-(1-methylpropyl)-3H-1,2,4-triazol-3-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-31 to 2018-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris Cedex, July 2010.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, May 2008, including most recent amendments.

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-(butan-2-yl)-4-{4-[4-(4-methoxyphenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one

EC Number:

607-685-0

Cas Number:

252964-68-4

Molecular formula:

C23H29N5O2

IUPAC Name:

1-(butan-2-yl)-4-{4-[4-(4-methoxyphenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-7982559-AAA (T001329)
- Physical state: solid (powder)
- Appearance: Grey-brown powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I14IB3639
- Expiration date of the lot/batch: 17 September 2020 (retest date)
- Purity/composition correction factor: 1
- Purity: 97.9% w/w (calculated as is) - HPLC

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: There was no information available regarding the solubility or stability in vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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 was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

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: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Aproximately 10 weeks old
- Weight at study initiation: 18.2 - 21.9 grams
- Housing: Group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6 of the main study, 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 (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 (deg C): 18 to 24°C; actual daily mean temperature during the study period: 22°C
- Humidity (%): 40 to 70%; actual main relative humidity during the study period: 40 to 43%
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

PreScreen Test: 25% and 50% w/w
Main Study: 0, 2%, 25%, 50% w/w

No. of animals per dose:

Five females per group; 4 groups (including control group)

Details on study design:

Rationale vehicle selection:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at the testing facility. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v) (turbid solution), N,N-dimethylformamide (not tested according to vehicle selection criteria), methylethylketone (not tested according to vehicle selection criteria), propylene glycol (not tested according to vehicle selection criteria) and dimethylsulfoxide (not tested according to vehicle selection criteria).

PRE-SCREEN TESTS:
- Compound solubility: no data
- Irritation: No irritation was observed in any of the animals examined. White/lilac test remnants were present on the dorsal surface of the ears of the animals between Days 1 and 4, which did not hamper scoring of the ears.
- Systemic toxicity: No signs of systemic toxicity were observed in any of the animals examined.
- Ear thickness measurements: 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 concentration selected for the main study was a 50% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
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:
SI value UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skinreaction
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 data

Results and discussion

Positive control results:

At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 2.2, and 3.5 respectively. An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was in the accepable range of 4.8 and 19.5%. The results of the 6 monthly reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%
Based on the results, it was concluded that the Local Lymph Node Assay as performed in the laboratory is an appropriate model for testing contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.7

Variability:

+/- 0.6

Test group / Remarks:

Based on 5 animals in 2% w/w in acetone/olive oil 4:1 group

Results 2

Parameter:

SI

Value:

1.5

Variability:

+/- 0.2

Test group / Remarks:

Based on 5 animals in 25% w/w in acetone/olive oil 4:1 group

Results 3

Parameter:

SI

Value:

4

Variability:

+/- 0.5

Test group / Remarks:

Based on 5 animals in 50% w/w in acetone/olive oil 4:1 group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 973 ± 138
2% w/w group: mean DPM ± SEM: 1647 ± 541
10% w/w group: mean DPM ± SEM: 1418 ± 210
25 %w/w group: mean DPM ± SEM: 3935 ± 483
SEM = Standard Error of the Mean

EC3 CALCULATION
An EC3 value (the estimated test item concentration that will give a SI =3) of 40% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation:
No irritation of the ears was observed in any of the animals examined. Lilac test item remnants were present on the dorsal surface of the ears of all animals treated at 25 and 50% between Days 1 and 3, which did not hamper scoring of the skin reactions.
- Systemic toxicity:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
- Macroscopy of the auricular lymph nodes and surrounding area:
The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals of the highest dose group, which were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The results of this study indicate that the test item could elicit a SI ≥ 3. An EC3 value (the estimated test item concentration that will give a SI =3) of 40% was calculated.
Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), JNJ-7982559-AAA (T001329) would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), JNJ-7982559-AAA (T001329) should be classified as skin sensitizer (Category 1B).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), JNJ-7982559-AAA (T001329) should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
.