(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th May 2020 to 10th June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In-vivo testing was conducted for other non EU regulatory requirements

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

other: EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

EC Number:

840-203-9

Cas Number:

2295746-51-7

Molecular formula:

C21H35N3O3

IUPAC Name:

(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 20.3 to 26.6 g.
- 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 (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier &Söhne GmbH+ CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled.

- Diet: Pelleted rodent diet(SMR/M-Z from SSNIFF® Spezialdiäten GmbH,Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap-water ad libitum
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 40 - 50%
- Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod: (12 hrs dark / 12 hrs light)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Three test item concentrations were tested; a 10%, 25% and 35% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.

No. of animals per dose:

Five animals / group

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. Two test item concentrations were tested; a 25% and 35% concentration. The highest concentration was the highest concentration that could be prepared homogeneously. The test system, procedures and techniques were identical to those used in the main study except that a 35% concentration was applied using a pipette with the tip cut off, that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. 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.

MAIN STUDY
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-methylthymidine. After five hours, all animals were euthanized. 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 (maze size: 200 µm, diameter: ± 1.5 cm). 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).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The SI values calculated for Alpha- Hexylcinnamaldehyde, technical grade (HCA). The test item concentrations 5, 10 and 25% were 2.4, 2.9 and 4.5, respectively. An EC3 value of 10.9% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at
Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

> 35

Test group / Remarks:

35% (w/w)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10% (w/w)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

25% (w/w)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 4

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

35% (w/w)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

Skin Reactions / Irritation
The very slight irritation of the ears as shown by all animals treated at 35% between Days 2 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 25% and 35% on Days 1-5, 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. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals of the test item and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 35% were 1038, 919 and 984 DPM, respectively. The mean DPM/animal value for the vehicle control group was 651 DPM. The SI values calculated for the test item concentrations 10, 25 and 35% were 1.6, 1.4 and 1.5, respectively.

Terminal procedures
No necropsy was performed, since all animals survived until the end of the observation period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits an SI ≥ 3 when tested up to 35%, PF-07202371-S7 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give an SI=3) (if any) exceeds 35%.

Based on these results, PF-07202371-S7 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 (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether PF-07202371-S7 induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.

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.
Test item concentrations selected for the main study were based on the results of  pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 35% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methylthymidineand 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.
Mean DPM/animal values for the test item groups treated with test item  concentrations 10, 25 and 35% were 1038, 919 and 984 DPM, respectively. The mean DPM/animal value for the vehicle control group was 651 DPM. The SI values calculated for the test item concentrations 10, 25 and 35% were 1.6, 1.4 and 1.5, respectively.
Since there was no indication that the test item elicits an SI ≥ 3 when tested up to 35%, PF07202371-S7 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give an SI=3) (if any) exceeds 35%.
Based on these results, PF-07202371-S7 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 (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).