(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th May 2019 to 28th May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EURL ECVAMDB-ALMProtocol n° 155: KeratinoSensTM. (Adopted March, 2018)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

1

Reference substance name:

(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

IUPAC Name:

(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

In vitro test system

Details on the study design:

A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSensTM cell line). The KeratinoSensTM cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).
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+9 in experiment 1, P+22 in experiment 2 and P+3 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 ± 1 hat 37±1.0oC in the presence of 5% CO2. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. 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) 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 5 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)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570nmwith the TECAN Infinite® M200 Pro Plate Reader.

Results and discussion

Positive control results:

The EC1.5 of the positive control, Ethylene dimethacrylate glycol, was within two standard deviations of the historical mean (125 µM, 59 µM and 7.4 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.39-fold, 2.55-fold and 6.65-fold in experiment 1, 2 and 3, respectively).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Run 3 (48h) 23 – 2000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: PF-04965842-01 showed toxicity. The calculated IC30 could not be calculated accurately since there was a biphasic response and the calculated IC50 was 1135 µM. • A dose related luminescence activity induction was observed after treatment with PF-04965842-

Results 2

Key result

Run / experiment:

other: Run 2 (48 h) 0.98 - 2000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: PF-04965842-01 showed toxicity. The calculated IC30 was 568 µM and the calculated IC50 was 808 µM. • A dose related luminescence activity induction was observed after treatment with PF-04965842-01. The Imax was 4.15 and the EC1.5 66 µM.

Results 3

Key result

Run / experiment:

other: Run 1 (48hrs) 0.98 - 2000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: PF-04965842-01 showed toxicity. The calculated IC30 was 30 µM and the calculated IC50 was 532 µM. • An induction was observed after treatment with PF-04965842-01. The Imax was 5.68 and the EC1.5 333 µM.

Applicant's summary and conclusion

Interpretation of results:

other: classified as positive in the KeratinoSensTM assay

Conclusions:

PF-04965842-01 is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control in two out of three experiments.

Executive summary:

The objective of this study was to evaluate the ability of PF-04965842-01 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens^TM assay. The study procedures described in this report were based on the most recent OECD guideline. Batch 19-AN-00220 of PF-04965842-01 was a white powder.PF-04965842-01 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) in the first two experiments and resulting in test concentrations of 23 – 2000 µM (1.5-fold dilution series) in the third experiment. The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed.
All experiments passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene
dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (125 µM, 59 µM and 7.4 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.39-fold, 2.55-fold and 6.65-fold in experiment 1, 2 and 3, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.3%, 3.5% and 4.0% in experiment 1, 2 and 3 respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
In the first experiment,PF-04965842-01 showed toxicity (IC30 value of 30 µM and an IC50 value of 532 µM). An induction of the luciferase activity (EC1.5 value of 333 µM) was measured. The maximum luciferase activity induction (Imax) was 5.68-fold leading to an individual run conclusion of negative since a positive result (>1.5-fold induction) was observed at test concentrations with a cell viability of <70% compared to the vehicle control.
In the second experiment,PF-04965842-01 showed toxicity (IC30 value of 568 µM and an IC50 value of 808 µM). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 66 µM) was measured. The maximum luciferase activity induction (Imax) was 4.15-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.
Since the first two experiments were not concordant a third experiment was performed to provide a final conclusion.

In the third experiment,PF-04965842-01 showed toxicity (IC30 value could not be calculated accurately since there was a biphasic response and an IC50 value of 1135 µM). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 55 µM) was measured. The maximum luciferase activity induction (Imax) was 7.23-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.
In conclusion, PF-04965842-01 is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control in two out of three experiments.