Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 2016 to 28 June 2016
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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.429, "Skin Sensitization: Local Lymph Node Assay", Paris, July 2010.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B42: "Skin Sensitization: Local Lymph Node Assay". Official Journal of the European Union No. L142, May 2008, including most recent
amendments.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS
870.2600. “Skin Sensitization”, March 2003.
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity (by HPLC @ 225 nm) 100.0 % [a/a]

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Species Mouse, CBA/J strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals 20 females (nulliparous and non-pregnant), five females per group (main study only).
Age and body weight Young adult animals (approx. 10 weeks old) were selected.
Body weight variation was within +/- 20% of the sex mean.
Identification Tail mark with a marker pen.
Health inspection At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact an d free from any abnormality.
Reliability check The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materi als, animal supplier, animal strain and essential procedures are summarized in APPENDIX 2 of this
report. For both scientific and animal welfare reasons, no concurrent positive control group was includ ed in the study. An extensive data base is available with reliability checks performed at half year inter vals during at least the past 9 years showing reproducible and consistent positive results.

5.3. Animal Husbandry
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
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. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. 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
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
(Merck, Darmstadt, Germany)
Concentration:
10, 25, 50%
No. of animals per dose:
5 females
Details on study design:
5.5. 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 (see section 0) 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.

The test system, procedures and techniques were identical to those used in the main study except that the application method may have been different (see tables) and 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.

5.6. Main Study
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 the vehicle.

5.6.1. Allocation
Group animal numbers induction (test item; % w/w)
1 01 - 05 0 (Dimethyl sulphoxide)
2 06 - 10 10
3 11 - 15 25
4 16 - 20 50

5.6.2. Induction - Days 1, 2 and 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. 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.

5.6.3. 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.4

5.6.4. Tissue Processing for Radioaáivity - 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.

5.6.5. Radioaáivity 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).

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.3
Variability:
± 0.6
Test group / Remarks:
10% Concentration
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Parameter:
SI
Value:
2
Variability:
± 1.0
Test group / Remarks:
25% Concentration
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Parameter:
SI
Value:
1.4
Variability:
± 0.7
Test group / Remarks:
50% Concentration
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction

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 3 when tested up to 50%, PF-06478031-01 was not considered to be a skin sensitizer.

Based on these results, PF-06478031-01 would not be 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 items and mixtures (including all amendments).