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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jun 2018 to 26 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
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
Details on test material:
Off-white powder
Specific details on test material used for the study:
Purity/Composition: 98.7% Test item storage: At room temperature protected from light Stable under storage conditions until: 20 November 2019 (retest date) (taken from label)

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 11 weeks old) were selected. Weight at the Initiation of Dosing: 20.6 to 25.6 g.

Justification for Test System and Number of Animals The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHW). The test method and number of animals were based on the test guidelines. 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 materials, animal supplier. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results. The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study. 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. The disposition of all animals was documented in the study records.

Husbandry
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 sawdust 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.

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 to 23°C with an actual daily mean relative humidity of 43 to 70%. 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 (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25% and 50% test item concentration, no to very slight erythema and no signs of toxicity were noted. Variation in ear thickness during the observation period slightly exceeded 25% from Day 1 pre-dose values for one animal at 50% on Day 6. Since the results of the ear thickness measurements were not conclusive, 50% was selected as the highest concentration to be used in the main study and ear thickness measurements were added to the main study for the 50% concentration.
No. of animals per dose:
5f/group
Details on study design:
Justification of Route and Dose Levels
Dose route and dose concentrations used are in compliance with the OECD test guidelines for LLNA studies.

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. Two test item concentrations were tested; a 25% and 50% 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 the animals were approximately 10 weeks (at initiation of treatment), 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.

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.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) 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) (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 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) (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 (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).

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Postdose Observations
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

Body Weights
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

Irritation
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). According to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded

Ear Thickness measurements
Ear thickness measurements were performed for all animals treated at 50% prior to dosing on Days 1 and 3, and on Day 6 using a digital thickness gauge (Kroeplin C110T-K).

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

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. DPM values are presented for each animal and for each dose group. 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 the DPM/vehicle control group mean. 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 (2017) (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 items and mixtures, including all amendments.
Positive control substance(s):
other: no concurrent positive control group will be 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.

Results and discussion

Positive control results:
no concurrent positive control group will be included in the study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 1.6
Test group / Remarks:
10 % test group
Key result
Parameter:
SI
Value:
ca. 1.5
Test group / Remarks:
25 % test group
Key result
Parameter:
SI
Value:
ca. 1.5
Test group / Remarks:
50 % test group
Cellular proliferation data / Observations:
Pre-screen Test
At a 25% and 50% test item concentration, no to very slight erythema and no signs of toxicity were noted. Variation in ear thickness during the observation period slightly exceeded 25% from Day 1 pre-dose values for one animal at 50% on Day 6. Since the results of the ear thickness measurements were not conclusive, 50% was selected as the highest concentration to be used in the main study and ear thickness measurements were added to the main study for the 50% concentration.

Skin Reactions / Irritation
The very slight erythema as noted for the animals treated at 25% and 50% between Days 2 and 6 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 the animals treated at 25% and 50% between Days 1 and 4, which did not hamper scoring of the skin reactions.

Ear Thickness
Variation in ear thickness during the observation period were less than 25% from Day 1 predose values for all animals.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. 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
The majority of auricular lymph nodes were considered normal in size, except for the nodes of three animals treated at 10%, two animals treated at 25% and two animals treated at 50%, which were considered to be enlarged. 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 50% were 911, 834 and 833 DPM, respectively. The mean DPM/animal value for the vehicle control group was 557 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.6, 1.5 and 1.5, respectively.

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 50%, PF06932437 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 50%. 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. Based on these results, PF-06932437 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-06932437 induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this study plan. The study was carried out based on the guidelines described in:   OECD, Section 4, Health Effects, No.429 (2010),   EC No 640/2012, Part B:  "Skin Sensitization: Local Lymph Node Assay"  EPA, OPPTS 870.2600 (2003) “Skin Sensitization”. Test item concentrations selected for the main study were based on the results of a pre-screen test.  At a 25% and 50% test item concentration, no to very slight erythema and no signs of toxicity were noted.  Variation in ear thickness during the observation period slightly exceeded 25% from Day 1 pre-dose values for one animal at 50% on Day 6.  Since the results of the ear thickness measurements were not conclusive, 50% was selected as the highest concentration to be used in the main study and ear thickness measurements were added to the main study for the 50% concentration.

In the main study, three experimental groups of five female CBA/J mice were treated with test item 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 the vehicle alone (N,N-dimethylformamide).  Three days after the last exposure, all animals were injected with 3H-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.

The very slight erythema as noted for the animals treated at 25% and 50% between Days 2 and 6 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 the animals treated at 25% and 50% between Days 1 and 4, which did not hamper scoring of the skin reactions. Variation in ear thickness during the observation period were less than 25% from Day 1 predose values for all animals.  No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.  Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.   The majority of auricular lymph nodes were considered normal in size, except for the nodes of three animals treated at 10%, two animals treated at 25% and two animals treated at 50%, which were considered to be enlarged.  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 10, 25 and 50% were 911, 834 and 833 DPM, respectively.  The mean DPM/animal value for the vehicle control group was 557 DPM.  The SI values calculated for the test item concentrations 10, 25 and 50% were 1.6, 1.5 and 1.5, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 50%, PF06932437 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 50%. 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. Based on these results, PF-06932437 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).