Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

In two Local Lymph Node Assays (LLNA) in mice (CBA/J resp. CBA/Ca) according to OECD Guideline 429 and EU Method B.42, the substance T003422 was not considered a skin sensitiser (Latour, 2017 and Moore, 2012).

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (initiated before October 11th 2016) is available.

Respiratory sensitization:

No reliable respiratory sensitisation study was available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-07-20 to 2016-08-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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 information:
Composition 1
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., I15AB0305
- Expiration date of the lot/batch: 23 January 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 formulations

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.


Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: young adults (approx. 10 weeks old)
- Weight at study initiation: 20.3 - 24.5 g
- 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 Spezialdiaten 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%, study plan deviation: deviations from the maximum level of daily mean relative humidity occurred. Evaluation: laboratory historical data do not indicate an effect of the deviations. The study integrity was not affected by the deviation.
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
methyl ethyl ketone
Concentration:
pre-screen test: 25 and 50% w/w
main study: 0, 2, 25 and 50% w/w
No. of animals per dose:
5 females per group; 4 groups
Details on study design:
PRE-SCREEN 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 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.
- Irritation: very slight erythema noted for all animals between Days 1 and 3. Scaliness was noted for all animals between Days 4 and 6.
- Systemic toxicity: no signs of systemic toxicity 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. White test item remnants, present on the dorsal surface of the ears of all animals (between Days 1 and 5), did not hamper scoring of the skin reactions.
Based on these results, the highest test concentration selected for the main study was a 50% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT
- 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 only.
TREATMENT
INDUCTION days 1, 2, 3:
- 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 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).

- 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 UN-GHS 2015; 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 data
Positive control results:
At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 1.5, and 4.3 respectively. An EC3 value of 18.0% 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 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 in the laboratory is an appropriate model for testing contact hypersensitivity.
Parameter:
SI
Value:
1
Variability:
+/- 0.2
Test group / Remarks:
Based on 5 animals in methyl ethyl ketone only group
Parameter:
SI
Value:
1.1
Variability:
+/- 0.3
Test group / Remarks:
Based on 5 animals in 2% w/w in methyl ethyl ketone group
Parameter:
SI
Value:
1.3
Variability:
+/- 0.3
Test group / Remarks:
Based on 5 animals in 25% w/w in methyl ethyl ketone group
Parameter:
SI
Value:
1.4
Variability:
+/- 0.4
Test group / Remarks:
Based on 5 animals in 50% w/w in methyl ethyl ketone group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% w/w group: mean DPM/animal: 441 ± 72
2% w/w group: mean DPM/animal: 504 ± 91
25% w/w group: mean DPM/animal: 563 ± 113
50 %w/w group: mean DPM/animal: 612 ± 149

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the item concentrations 2, 25 and 50% were 1.1, 1.3 and 1.4, respectively. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, T003422 was not considered to be a skin sensitizer.

EC3 CALCULATION
not applicable for this test (SI values < 3)

CLINICAL OBSERVATIONS:
- Skin reactions/irritation:
No erythema of the ears was observed in any of the animals examined. The scaliness of the ears as shown by several control animals between Days 3 and 5 was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of all animals at a concentration of 25% and 50% (between Days 1 and 6), 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 out of five animals treated at 50%, where were considered slightly smaller. 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.
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%, T003422 was not considered to be a skin sensitizer.

Based on these results, T003422 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 (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a key LLNA study by Latour (2017), three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 25 or 50% w/w for three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No erythema of the ears was observed in any of the animals examined. The scaliness of the ears as shown by several control animals between Days 3 and 5 was considered not to have a toxicologically significant effect on the activity of the nodes.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in two out of five animals treated at 50%, which were considered slightly smaller. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 25 and 50% were 504, 563 and 612 DPM, respectively. The mean DPM/animal value for the vehicle control group was 441 DPM. The SI values calculated for the item concentrations 2, 25 and 50% were 1.1, 1.3 and 1.4, respectively.

Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, T003422 was not considered to be a skin sensitizer.

In a second supporting (non-GLP) LLNA study, Moore (2012) investigated the skin sensitisation potential of T003422 following topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25 and 50% (w/v) in acetone:olive oil (4 +1).

Preliminary investigations were performed at 25 and 50% w/v with 2 female mice per concentration to establish the highest concentration of test substance which did not lead to systemic toxicity or excessive local irritation.

The main phase of the study comprised three treated groups, each comprising four female mice receiving JNJ-42808415 at concentrations of 10, 25 or 50% w/v.

Similarly constituted groups received the vehicle (acetone:olive oil (4:1 w/v)) or positive control substance (25% v/v hexyl cinnamic aldehyde). The mice were treated by daily application of 25ml of the appropriate concentration or control(vehicle or positive), to the dorsal surface of both ears for three consecutive days.

The SI obtained for 10, 25 and 50% w/v were 1.2, 1.5 and 1.1, respectively, which indicated that JNJ-42808415 did not show the potential to induce skin sensitization.

The EC3 (Estimated Concentration of Three) value was considered to be greater than the highest concentration tested (50% w/v). The SI for the positive control substance hexyl cinnamic aldehyde was 5.9.

T003422 was not regarded as a potential skin sensitizer.

 

Waiver in vitro skin sensitisation study:

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (initiated before October 11th 2016) is available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

Based on two LLNA studies in female mice (Latour 2017 and Moore 2012), the test item does not have to be classified under EU CLP.

Respiratory sensitisation:

No data were available to decide on the classification for respiratory sensitisation.