Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May - 20 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008 Part B. Skin Sensitization: Local Lymph Node Assay, May 2008

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

EC Number:

816-314-3

Cas Number:

927881-65-0

Molecular formula:

C83H87N3O21

IUPAC Name:

Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

Test material form:

solid: particulate/powder

Details on test material:

White solid
Test item storage: At room temperature

Specific details on test material used for the study:

White Solid
Test item storage: At room temperature
Batch: 902/6460031/D/13/1 (is same batch as batch E010017351 on Certificate of Analysis)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 10 weeks old) were selected.
Weight at the Initiation of Dosing: 18.8 to 24.0 g.
Animal Identification:
At study assignment, each animal was identified using a tail mark with indelible ink.
Environmental Acclimation:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
Housing:
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles. Animals were separated during designated procedures/activities. Each cage was clearly labeled.
Environmental Conditions:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 42 to 65%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Food:
Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures.
Water
Municipal tap-water was freely available to each animal via water bottles.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Test item concentrations of 2, 5 or 10% w/w was administered on three consecutive days

No. of animals per dose:

Three experimental groups of five female CBA/J mice. One group of five animals was treated with the vehicle.

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.
Initially, Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-14 weeks (at initiation of treatment) 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. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.
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.

Allocation

Group animal numbers induction (test item; % w/w)
1 01 - 05 0 (AcOO)
2 06 - 10 2
3 11 - 15 5
4 16 - 20 10

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.
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 μCi 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 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. 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) 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 (2910TR). 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

Positive control results:

No concurrent positive control group was included in the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

2% concentration

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

5% concentration

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

10% concentration

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Any other information on results incl. tables

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 786, 1071 and 590 DPM, respectively. The mean DPM/animal value for the vehicle control group was 596 DPM.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, PF-06460260 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Executive summary:

Based on these results, PF-06460260 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).