cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

Endpoint summary

Description of key information

A DEREK assessment yielded no alerts for skin and therefore  PF-07085579 was predicted negative. However as a substructure was not recognized within the prediction domain of DEREK the negative prediction has some uncertainty. A DPRA could not be performed as the substance did not dissolve in a suitable solvent. The KeratinoSensTM assay was negative as no biologically relevant activation of keratinocytes was observed at non-cytotoxic concentrations. Performance of the DPRA and KeratinoSensTM assay revealed that PF07085579 is difficult to solubilize in various aqueous solvents and DMSO. Furthermore, the substance was observed to have cytotoxic properties at low concentrations. Difficulties in test item preparation and cytotoxicity were therefore expected when performing Step 2 (a USENS TM assay). Occurrence of cytotoxicity and/or precipitation will result in a positive outcome disregarding potential absence of activation of dendritic cells at non-cytotoxic concentrations.

For this reason, results from a U-SENS TM assay was not considered as it would not aid in a conclusive result on skin sensitization. As further in vitro testing is scientifically not justified it was considered necessary to perform in vivo testing to determine the skin sensitizing properties of  PF07085579. A LLNA study was performed and since there was no indication that the test item elicits a  SI  ≥ 3 when tested up to 25%,  PF07085579 was not considered to be a skin sensitizer.

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:

5th Feb 2019 to 25th Feb 2019

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)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

20 Females (nulliparous and non-pregnant). Five females per group. Age at the Initiation of Dosing: Young adult animals (approximately 12 weeks old) were selected.
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 47%. 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.

Vehicle:

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

Concentration:

0%, 5%, 10%, 25%

No. of animals per dose:

5

Details on study design:

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.

Key result

Parameter:

SI

Value:

ca. 1

Variability:

+/- 0.1

Test group / Remarks:

0%

Remarks on result:

other: Vehicle Control

Key result

Parameter:

SI

Value:

ca. 1.2

Variability:

+/-0.3

Test group / Remarks:

5%

Remarks on result:

not determinable

Remarks:

No indication of skin sensitisation was detected

Key result

Parameter:

SI

Value:

ca. 1.2

Variability:

+/-0.1

Test group / Remarks:

10%

Remarks on result:

not determinable

Remarks:

No indication of skin sensitisation was detected

Key result

Parameter:

SI

Value:

ca. 0.9

Variability:

+/-0.1

Test group / Remarks:

25%

Remarks on result:

not determinable

Remarks:

No indication of skin sensitisation was detected

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 25%, PF07085579 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 25%. 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-07085579 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:

23rd Nov 2018 to 14th Dec 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)

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSensTM. (Adopted March, 2018).

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Two independent experiments were performed. The cells were in these experiments
incubated with PF-07085579 in a concentration range of 0.49 – 1000 µM (2-fold dilution
series) in the first experiment and in test concentrations of 11 – 125 µM (1.25-fold dilution
series) in the second experiment for 48 hours ± 1 h. The activation of the ARE-dependent
pathway was assessed by measuring the luminescence induction compared to the vehicle
control. In addition, the viability was assessed with an MTT assay.

Key result

Run / experiment:

other: 0.49 – 1000 µM

Parameter:

other: IC30

Value:

85 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 0.49 - 1000 µM

Parameter:

other: IC50

Value:

115 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 0..49 - 1000 µM

Parameter:

other: EC1.5

Remarks:

nduction of the luciferase activity

Value:

97 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 11 - 125 µM

Parameter:

other: IC30

Value:

122 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In the first experiment, PF-07085579 showed toxicity (IC30 values of 85 µM and an IC50 value of 115 µM). An induction of the luciferase activity (EC1.5 value of 97 µM) was measured and the maximum luciferase activity induction (Imax) was 2.37-fold. Since the induction was observed at cytotoxic concentrations and not statistically significant the individual run conclusion was negative. In the second experiment, PF-07085579 showed toxicity (IC30 values of 122 µM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 0.75-fold and the individual run conclusion was negative as well. PF-07085579 is classified as negative in the KeratinoSensTM assay since negative results were observed at test concentrations with cell viability >70% and was tested up to or beyond the cytotoxic level.

In conclusion, PF-07085579 is classified as negative (no biologically relevant 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-07085579 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline. Batch GR13449 of  PF-07085579 was a white solid.  PF-07085579 was dissolved in dimethyl

sulfoxide at 100 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 0.49 – 1000 µM (2-fold dilution series) in the first experiment. The highest test concentration was considered to be the limit of solubility. In the second experiment, a more narrow dose response analysis was performed using a lower dilution factor of 1.25-fold to investigate the induction at 125 µM in experiment 1 in more detail.

No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both 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 (67 µM and 98 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.41-fold and 2.20-fold in experiment 1 and 2, respectively).

• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (15% and 4.7% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

In the first experiment,  PF-07085579 showed toxicity (IC30 values of 85 µM and an IC50 value of 115 µM). An induction of the luciferase activity (EC1.5 value of 97 µM) was measured and the maximum luciferase activity induction (Imax) was 2.37-fold. Since the induction was observed at cytotoxic concentrations and not statistically significant the individual run conclusion was negative. In the second experiment,  PF-07085579 showed toxicity (IC30 values of 122 µM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 0.75-fold and the individual run conclusion was negative as well.  PF-07085579 is classified as negative in the KeratinoSensTM assay since negative results were observed at test

concentrations with cell viability >70% and was tested up to or beyond the cytotoxic level. In conclusion,  PF-07085579 is classified as negative (no biologically relevant 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, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

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))

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Activation and maturation of dendritic cells; OECD 442E Myeloid U937 Skin Sensitization Test (U-SENS TM )

Qualifier:

no guideline required

Guideline:

other: DEREK assessment

GLP compliance:

yes

Type of study:

other: DEREK assessment, Direct Peptide Reactivity Assay, KeratinoSensTM assay, U-SENS test

Species:

mouse

Strain:

CBA

Sex:

not specified

Parameter:

other:

Remarks:

DEREK assessment yielded no alerts for skin and therefore PF-07085579 was predicted negative. DPRA could not be performed. Keratinocytes assay was negative. LLNA test required to determine skin sensitizing properties

Value:

0

Remarks on result:

not determinable

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

No indication of skin sensitisation was detected

Interpretation of results:

study cannot be used for classification

Conclusions:

A DEREK assessment yielded no alerts for skin and therefore PF-07085579 was predicted negative. However as a substructure was not recognized within the prediction domain of DEREK the negative prediction has some uncertainty. A DPRA could not be performed as the substance did not dissolve in a suitable solvent. The KeratinoSensTM assay was negative as no biologically relevant activation of keratinocytes was observed at non-cytotoxic concentrations. Performance of the DPRA and KeratinoSensTM assay revealed that PF07085579 is difficult to solubilize in various aqueous solvents and DMSO. Furthermore, the substance was observed to have cytotoxic properties at low concentrations. Difficulties in test item preparation and cytotoxicity are therefore expected when performing Step 2 (a USENS TM assay). Occurrence of cytotoxicity and/or precipitation will result in a positive outcome disregarding potential absence of activation of dendritic cells at non-cytotoxic concentrations.
For this reason, results from a U-SENS TM assay will not aid in a conclusive result on skin sensitization. As further in vitro testing is scientifically not justified as it will not lead to a final conclusion on presence or absence of skin sensitizing properties, it is recommended to perform in vivo testing to determine the skin sensitizing properties of PF07085579.

Executive summary:

The 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 and in vitro data. A DEREK assessment, DPRA and KeratinoSens TM assay were initiated 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 DEREK prediction on the skin sensitizing potential of  PF-07085579 was negative, however as a substructure was not recognized within the prediction domain of DEREK the prediction has some uncertainty. A DPRA could not be performed as the substance did not dissolve in a

suitable solvent. The KeratinoSensTM assay did not show a biologically relevant activation of keratinocytes when exposed to  PF-07085579 at non-cytotoxic concentrations and was therefore negative. Due to its limited solubility, difficulties in test item preparation and cytotoxicity are expected during the performance of a U-SENS TM assay. The outcome of such study is not expected to aid in the overall conclusion on skin sensitization for  PF-07085579. Since the current data-set is insufficient to conclude on skin sensitizing potential of  PF07085579 and further in vitro testing is not expected to yield information that allows a final conclusion on this endpoint, it is scientifically justified to omit further in vitro testing and to proceed with in vivo testing.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available