4-(4-bromo-3-formylphenoxy)benzonitrile

Endpoint summary

Description of key information

A DEREK assessment, DPRA assay and KeratinoSensTM assay were performed:.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for  PF06932437.

In the DPRA assay, the test item did not show significant binding to cysteine and/or lysine moieties.However, since precipitation of the test item was seen during the test, this negative result is uncertain and should be interpreted with caution.

In the KeratinoSens assay,  PF-06932437 did not activate the anti-oxidant/electrophile responsive element (ARE) dependent pathway in keratinocytes) in a KeratinoSens

TM assay. Performance of a U-SENSTM assay was omitted, as cytotoxicity and precipitation are expected at test concentrations, which would lead to an inconclusive or positive result.

Taking all data together, the in silico, in chemico and in vitro data do not allow final

conclusion on the skin sensitizing properties of  PF-06932437. It is therefore recommended to

perform an in vivo test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

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)

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)

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.

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.

Positive control results:

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

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.

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).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Feb 2018 to 09 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Principles of method if other than guideline:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is proposed to address this second key event. Skin sensitizers have been reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes. In the KeratinoSensTM cell line activation of the Nrf2 pathway results in luciferase expression which can be quantified easily. The design of this study is based on the following study guideline:  OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

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)

Details on the study design:

Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+16 in experiment 1, P+18 in experiment 2 and P+4 in experiment 3.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. In total 3 valid experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

All experiments passed the acceptance criteria: The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 µM (122 µM, 63 µM and 80 µM in experiment 1, 2 and 3, respectively). A dose response was observed in all experments and the induction at 250 µM was higher than 2-fold in 2 out of 3 experiments (2.20-fold and 2.14-fold in experiment 1 and 3, respectively). Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.8%, 11.9% and 3.9% in experiment 1, 2 and 3, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 uM

Remarks:

induction of the luciferase activity

Value:

72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: experiment 2 and 3

Parameter:

other: EC1.5 value

Remarks:

no EC1.5 value

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The KeratinoSensTM test is considered acceptable if it meets the following criteria: a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control. c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded

All experiments passed the acceptance criteria:  The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.  The EC1.5 of the positive control was between 5 and 125 µM (122 µM, 63 µM and 80 µM in experiment 1, 2 and 3, respectively). A dose response was observed in all experments and the induction at 250 µM was higher than 2-fold in 2 out of 3 experiments (2.20-fold and 2.14-fold in experiment 1 and 3, respectively).  Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.8%, 11.9% and 3.9% in experiment 1, 2 and 3, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, PF-06932437 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of PF-06932437 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study procedures described in this report were based on the most recent OECD guideline. Batch GR12302 (groten) / BFPB31711001 (suven) of PF-06932437 was an off-white powder.  A correction factor of 1.013 was used to correct for the purity (98.7%).  PF-06932437 was dissolved in dimethyl sulfoxide at 200 mM.  From this stock 11 spike solutions in DMSO were prepared.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of  0.98 - 2000 µM (2-fold dilution series).  The highest test concentration was the highest dose required in the current guideline.  The test item precipitated at dose levels of 250 µM and upwards.  Three independent experiments were performed. All experiments passed the acceptance criteria:  The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.    The EC1.5 of the positive control was between 5 and 125 µM (122 µM, 63 µM and 80 µM in experiment 1, 2 and 3, respectively).  A dose response was observed in all experiments and the induction at 250 µM was higher than 2-fold in 2 out of 3 experiments (2.20-fold and 2.14-fold in experiment 1 and 3, respectively).  Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.8%, 11.9% and 3.9% in experiment 1, 2 and 3, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.   PF-06932437 showed toxicity in all experiments (IC30 values of 153 µM, 93 µM and 27 µM and IC50 values of 180 µM, 117 µM and 59 µM in experiment 1, 2 and 3, respectively).  In the first experiment an induction of the luciferase activity was measured (EC1.5 value of  72 µM). In experiment 2 and 3 no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations.  The maximum luciferase activity induction (Imax) was 1.94-fold, 0.99-fold and 1.44-fold in experiment 1, 2 and 3, respectively.  PF-06932437 is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations up to 2000 µM. In conclusion, PF-06932437 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.  

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Mar 2018 to 10 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guideline for DPRA studies. Testing required in a stepwise manner.

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:

Details on the study design:

Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24 hours and 23.5 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation. The SPCC samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature. For the SPCL test item samples that showed phase separation, the lower phase was transferred into a new vial and subjected to HPLC-PDA analysis.

HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following systems: System 1 (used for Cysteine Reactivity Assay): - Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands) - MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands) - LC Column oven 300 (Thermo Scientific) - Surveyor PDA detector (Thermo Scientific)

System 2 (used for Lysine Reactivity Assay): - Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands) - HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands) - Column Oven #151006 (Grace, Worms, Germany) - Surveyor PDA detector (Thermo Scientific)

Positive control results:

The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 56.7% ± 1.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Key result

Run / experiment:

other: Test item

Parameter:

other: Mean of SPCC and SPCL depletion %

Value:

4.3

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The following criteria had to be met for a run to be considered valid: a) The standard calibration curve had to have an r2>0.99. b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM. e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%. The following criteria had to be met for a test item’s results to be considered valid: a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion. b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. PF-06932437 was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation/phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Executive summary:

The objective of this study was to determine the reactivity of PF-06932437 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL).  After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm.  SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and  non-sensitizers. The study procedures described in this report were based on the most recent OECD guideline. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate/phase separation was observed.

In the cysteine reactivity assay the test item showed 1.5% SPCC depletion while in the lysine reactivity assay the test item showed 7.1% SPCL depletion.  The mean of the SPCC and SPCL depletion was 4.3% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.  PF-06932437 was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.  However, since precipitation/phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides.  Consequently, this negative result is uncertain and should be interpreted with due care.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

Weight of Evidence based on in silico/in chemico/in vitro data.

Type of information:

(Q)SAR

Remarks:

Derek Nexus 6.0.1

Adequacy of study:

weight of evidence

Study period:

May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source

Justification for type of information:

Weight of Evidence based on in silico/in chemico/in vitro data.

Qualifier:

no guideline required

Principles of method if other than guideline:

A DEREK assessment, DPRA assay and KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA. Guidance on information requirements and chemical safety assessment Chapter R.7a.

GLP compliance:

not specified

Justification for non-LLNA method:

hTe objective of this study was to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including in silico/in chemico/in
vitro data. A DEREK assessment, DPRA assay and KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. A weight of evidence approach according to Annex XI, sections 1.21.5, to the REACH Regulation is used.

Run / experiment:

other: Derek Nexus 6.0

Parameter:

other: structural alerts

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: no known structural alerts for skin sensitizing properties.

Other effects / acceptance of results:

A DEREK NEXUS assessment revealed that PF-06932437 has no known structural alerts for
skin sensitizing properties.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available