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Description of key information

Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
20 November 2018
Reliability:
1 (reliable without restriction)
Justification for type of information:
The objective of the study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
not specified
Justification for non-LLNA method:
The objective of the study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.
Key result
Parameter:
other: Results from DEREK NEXUS analysis
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. PF-07094402 is predicted to be not sensitizing to the skin.
Endpoint:
skin sensitisation: in chemico
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
05 February to 07 February, 2019
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))
GLP compliance:
yes
Justification for non-LLNA method:
The objective of this study was to determine the reactivity of PF-07094402 towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in one of four classes of reactivity for supporting the discrimination between skin sensitizers and nonsensitizers. The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C.
Details on the study design:
Test Item Preparation:
No correction for the purity/composition of the test item was performed.

Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol (EtOH) and methanol (MeOH).

Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 44.7 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1550 µL MeOH after vortex mixing and 7 minutes of sonication to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

Any residual volumes were discarded.

Synthetic Peptide Containing Cysteine (SPCC) Preparation:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.6 mg of SPCC in 21.16 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

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. In addition, a RCcysCMeOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMeOH sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MeOH.

A SPCC calibration curve was then prepared.

Synthetic Peptide Containing Lysine (SPCL) Preparation:
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.6 mg of SPCL in 20.46 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

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. In addition, a RClysCMeOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMeOH sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MeOH.

A SPCL peptide calibration curve was prepared.

The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.

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. 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 analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
System 2 (used for Lysine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
All samples were analyzed according to the HPLC method.


Key result
Parameter:
other: DPRA
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The results of the cysteine reactivity assay for the test item indicated that for the CC sample, no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 209803/A-cys samples, the mean SPCC A220/A258 area ratio was 37.76. Since this was within the 33.90-41.43 range, this again indicated that there was no co-elution of the test item with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls CMeOH. The mean Percent SPCC Depletion for the test item was 4.3% ± 5.8%.

The results of the lysine reactivity assay for the test item indicated that for the CC sample, no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 209803/A-lys samples, the mean SPCL A220/A258 area ratio was 32.38. Since this was within the 28.22-34.50 range, this again indicated that there was no co-elution of the test item with SPCL. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls CMeOH. The mean Percent SPCL Depletion for the Test Item was 2.0% ± 0.8%.

Upon preparation of the SPCC test item samples, no precipitate or phase separation was observed, however, after incubation a precipitate was observed. Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 4.3% SPCC depletion while in the lysine reactivity assay the test item showed 2.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 3.2% and as a result the test item 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.

At a concentration of 100 mM,  PF-07094402 was not soluble in ACN,  MQ, ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) and ethanol, but was soluble in methanol. Therefore, methanol was used to dissolve the test item in this DPRA study.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.
PF-07094402 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 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.
Endpoint:
skin sensitisation: in vitro
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
08 February - 22 March, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The objective of this study is to evaluate the ability of PF-07094402 to activate the antioxidant/ electrophile responsive element (ARE)-dependent pathway in the KeratinoSens(TM) assay.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes
Justification for non-LLNA method:
The objective of this study is to evaluate the ability of PF-07094402 to activate the antioxidant/ electrophile responsive element (ARE)-dependent pathway in the KeratinoSens(TM) assay.
Details on the study design:
Experimental 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 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.

Treatment of Cells
The medium was removed and replaced with fresh culture medium to which 50 µL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. In total 2 valid experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer and Steady-Glo Luciferase Assay Substrate from Promega 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 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.

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT 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 570 nm with the TECAN Infinite® M200 Pro Plate Reader
Key result
Parameter:
other: Results from KeratinoSens Assay
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, PF-07094402 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.
Endpoint:
skin sensitisation, other
Remarks:
WoE
Type of information:
other: DEREK, DPRA, KeratainoSens(TM)
Adequacy of study:
weight of evidence
Study period:
05 August 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Justification for type of information:
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, a DPRA and a 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.
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)
GLP compliance:
yes
Type of study:
other: A DEREK assessment, a DPRA and a KeratinoSensTM assay
Justification for non-LLNA method:
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.
Key result
Parameter:
other: Results from an in silico/in chemico/in vitro skin sensitization testing strategy
Remarks on result:
no indication of skin sensitisation

No data were available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment (Charles River project no 20171466), a DPRA (Charles River project no 20171467) and a KeratinoSensTM  assay (Charles River project no 20171468)

The DEREK NEXUS assessment was negative and predicted  PF-07094402 to be a non-sensitizer.

The DPRA was negative as no significant binding of the test item to cysteine- and lysine-residues was observed, however precipitation was observed and therefore the negative result should be interpreted with caution.

In the KeratinoSensTM  assay, no activation of keratinocytes was observed up to the highest concentration tested and therefore the result was negative. However precipitation and cytotoxicity was observed to occur at the highest test doses.

The combined negative results from the in vitro skin sensitization test strategy provide sufficient evidence to determine that  PF-07094402 does not have the potential to induce sensitization in humans.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results from an in silico/in chemico/in vitro skin sensitization testing strategy, it is concluded that PF-07094402 is not a skin sensitizer. PF-07094402 does not need to be classified for skin sensitization according to Regulation (EC) No 1272/2008 and related amendments.
Executive summary:

A DEREK assessment, a DPRA and a 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 inECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. The DEREK assessment was negative and predictedPF-07094402 to be a non-sensitizer. The DPRA was negative as no significant binding of the test item to cysteine- and lysine-residues was observed, however precipitation was observed and therefore the negative result should be interpreted with caution. In the KeratinoSensTM assay, no activation of keratinocytes was observed up to the highest concentration tested and therefore the result was negative. However precipitation and cytotoxicity was observed to occur at the highest test doses. The combined negative results from the in vitro skin sensitization test strategy provide sufficient evidence to determine thatPF-07094402 does not have the potential to induce sensitization in humans.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results from an in silico/in chemico/in vitro skin sensitization testing strategy, it is concluded that  PF-07094402 is not a skin sensitizer. PF-07094402 does not need to be classified for skin sensitization according to Regulation (EC) No 1272/2008 and related amendments.