Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 September - 28 September 2015
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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl 2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-1-thio--D-galactopyranoside
EC Number:
805-702-8
Cas Number:
322761-81-9
Molecular formula:
C45H48O10S
IUPAC Name:
ethyl 2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-1-thio--D-galactopyranoside
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: Yes. 20 females (nulliparous and non-pregnant), five females per group (main study only).
- Age at study initiation: Young adult animals (approx. 10 weeks old) were selected.
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- 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.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): A relative humidity of 40 to 70%.
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle
- IN-LIFE DATES: From: 16 September 2015 To: 28 September 2015

Study design: in vivo (non-LLNA)

No. of animals per dose:
5 females

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The mean DPM/animal value for the vehicle control group was 452 DPM.
No. of animals per dose:
In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).
Details on study design:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

Group animal numbers induction (test substance; % w/w)
1 01 - 05 0 (Acetone/Olive oil (4:1 v/v))
2 06 - 10 10
3 11 - 15 25
4 16 - 20 50

Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 NL/ear) with the test substance, 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 substance.

Excision of the nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 mCi of 3H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. 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 mm). 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 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 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).
Positive control substance(s):
other: For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
Statistics:
In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control. The methods used will be specified in the raw data and report.
The EC3 value (the estimated substance concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using linear interpolation (reference 1).
If it is not possible to determine the SI=3 value, additional groups of animals may be treated. This will be done in consultation with the Sponsor and will be confirmed by protocol amendment.

Results and discussion

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.2
Test group / Remarks:
substance concentration 10%
Parameter:
SI
Value:
1.1
Test group / Remarks:
substance concentration 25%
Parameter:
SI
Value:
1.8
Test group / Remarks:
substance concentration 50%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test substance elicited a SI >= 3 when tested up to 50%, PF- 06460246 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, PF-06460246 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance 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).