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Toxicological information

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:
From 2015-06-17 to 2015-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as stated in study reports): JNJ-4508530-AAA (T002707)
- Physical state: solid (powder)
- Appearance: white, beige powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Janssen Pharmaceutica N.V., I15BB0573
- Expiration date of the lot/batch: 08 February 2017 (retest date)
- Purity/composition correction factor: 1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Homogeneity was assessed by visual inspection of the solutions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item preparation were prepared with 4 hours prior to each dosing. Concentrations were stirred with a magnetic stirrer immediately prior to dosing.

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, source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19 - 24 grams
- Housing: animals were 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): free access to pelleted rodent diet (SM R/M-Z from SSNIFF ® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): free access to 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

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 2, 25 and 50% (w/w)
No. of animals per dose:
5 females per group; 4 groups
Details on study design:
PRE-SCREEN TEST:
- 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 as required in the test guidelines (50% for solids).
- 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.
- No irritation and no signs of systemic toxicity were observed in any of the animals examined. White staining of test item remnants on the dorsal surface of the ears of all animals between days 1 and 3, did not hamper scoring of erythema. 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
- Name of test method: Local Lymph Node Assay: Radioactivity measurements on day 7 were performed using a Packard scintillation counter (2800 TR). The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM)
- 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:
SI value < 3: No sensitizer
SI ≥ 3: Cat 1 Skin sensitizer (EC3 value ≤ 2%: sub-category 1A; EC3 value > 2%: sub-category 1B); Hazard statement: H317: May cause an allergic skin reaction

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item preparations (w/w) were prepared within 4 hours prior to each dosing.
- 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 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 3.0 and 9.1 respectively. An EC3 value of 10% 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:
2% group
Parameter:
SI
Value:
1.4
Test group / Remarks:
25% group
Parameter:
SI
Value:
1.2
Test group / Remarks:
50% group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% group: mean DPM/animal ± SEM: 693 ± 221
2% group: mean DPM/animal ± SEM: 835 ± 96
25% group: mean DPM/animal ± SEM: 979 ± 93
50% group: mean DPM/animal ± SEM: 819 ± 43

DETAILS ON STIMULATION INDEX CALCULATION
Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, JNJ-4508530-AAA (T002707) was not considered to be a skin sensitizer.

EC3 CALCULATION
not applicable (no SI values < 3)

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 of all animals treated at 25% and 50% between Days 1and 3, 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.

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.

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:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, T002707 was not considered to be a skin sensitizer. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Based on these results, T002707 was not regarded as a skin sensitizer according to the recommendations made in the test guidelines. 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 Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).