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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-27 to 2016-08-22
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-chlorophenyl cyclopentyl ketone
EC Number:
229-802-0
EC Name:
2-chlorophenyl cyclopentyl ketone
Cas Number:
6740-85-8
Molecular formula:
C12H13ClO
IUPAC Name:
2-chlorophenyl cyclopentyl ketone
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): JNJ-54582034-AAA (T003642)
- Physical state: liquid
- Appearance: yellow liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020709377
- Expiration date of the lot/batch: 2016-08-31 (Retest date)
- Purity/correction factor: 1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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. 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.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
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: 18.9 - 23.8 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, 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. Health inspection at least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
- Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were perfor
med according to facility standard procedures. There were no findings that could interfere with the
study.

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

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2%, 50% and 100% 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 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
- 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.

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 for three consecutive days. The concentrations were mixed wiwth a vortex mixer 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 An alytical 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 CountsPer Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.5 and 4.3 respectively. An EC3 value of 18% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.5, 14.5, 13.4, 14.1, 17.3 and 9.8%.
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.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.2
Test group / Remarks:
Based on 5 animals on 2% group
Parameter:
SI
Value:
9.6
Test group / Remarks:
Based on 5 animals on 50% group
Parameter:
SI
Value:
13.3
Test group / Remarks:
Based on 5 animals on 100% group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 50 and 100% were 374, 3070 and 4239 DPM, respectively. The mean DPM/animal value for the vehicle control group was 319 DPM.

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 2, 50 and 100% were 1.2, 9.6 and 13.3, respectively.

EC3 CALCULATION
The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 12.3% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: The very slight erythema of the ears as shown in all animals treated at 50% and 100% on Days 1, 2 and/or 3 and the scaliness as shown in all animals treated at 100% on Days 3, 5 and/or 6 were considered not to have a toxicologically significant effect on the activity of the nodes.
- Systemic toxicity: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Yellow/green discoloration of the urine was noted for all animals treated at 50% and 100% between Days 2 and 6 but not on Day 4. This was considered to be due to the colour of the test item but was considered not to have an impact on the results in the absence of any clinical signs.
- Macroscopy of the auricular lymph nodes and surrounding area: The majority of auricular lymph nodes of the animals treated at 2 and 50% were considered normal in size, except for one of the nodes in one animal treated at 50%, which were considered slightly enlarged. The node(s) of four animals treated at 100% were also considered slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Any other information on results incl. tables

Pre-screen test:

- At a concentration of 50% very slight erythema was noted for both animals on Days 2 and 3. At a concentration of 100% very slight to well-defined erythema was noted for both animals between Days 1 and 4, scabs were noted for both animals on Days 5 and/or 6 and quick breathing was noted for both animals on Day 3.

- 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 100% concentration.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The results of the study indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 12.3% was calculated.
According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), T003642 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), T003642 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.