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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Remarks:
Direct Peptide Reactivity Assay (DPRA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Mar 2018 to 05 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
OECD Guideline for the Testing of Chemicals, Guideline 442C. In Chemico Skin
Sensitization: Direct Peptide Reactivity Assay (DPRA) (4 February 2015)
Deviations:
no
GLP compliance:
yes
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)
Specific details on test material used for the study:
Purity 99.73%
Parameter:
other: mean Percent SPCC Depletion for the test item
Run / experiment:
Cysteine Reactivity Assay
Value:
ca. 66.5
Parameter:
other: mean Percent SPCL Depletion for the Test Item
Run / experiment:
Lysine Reactivity Assay
Value:
ca. 4.2
Parameter:
other: DPRA prediction and reactivity classification
Run / experiment:
Cysteine 1:10 / Lysine 1:50 prediction model
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, this DPRA test is valid. PF-06961030 was positive in the DPRA and was classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Apr 2018 to 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Purity/Composition: 99.73%
Details on study design:
4.5.1. Preparation of Test Item Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test item. All test item handlings were performed protected from light.

A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (light yellow solution). This stock was diluted to 100 mM (colourless solution). The 100-fold dilution in DMEM glutamax of 200 mM and 100 mM formed a clear solution. The 2000 μM (200 mM stock) concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).

In the main experiments the test item was dissolved/suspended in dimethyl sulfoxide (DMSO) at 200 mM (light brown to light yellow). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

No precipitation was observed at the start and end of the incubation period in the 96-well plates.

Test item concentrations were used within 3.5 hours after preparation.

Any residual volumes were discarded.

4.5.2. Preparation of the Positive Control
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.7.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

4.5.3. Preparation of the Vehicle Control
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

4.5.4. Blank
On each plate three blank wells were tested (no cells and no treatment).

4.6 Test System
Test System
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, Switserland). 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.

Rationale
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).

4.7 Cell Culture
Basic medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).

Exposure medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 66 – 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 – 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

4.8 Subculturing
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).

Experimental Design
4.9.1. 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+12 in experiment 1 and P+17 in experiment 2.

4.9.2. Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 mL culture medium containing serum but without Geneticin) to which 50 mL 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. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.

4.9.3. 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).

4.9.4. 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; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 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:
The EC1.5 of the positive control was between 5 and 125 μM (46 μM and 87 μM in experiment 1 and 2, respectively).Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
Parameter:
other: maximum luciferase activity induction (Imax)
Run / experiment:
Experiment 1
Value:
8.93
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: maximum luciferase activity induction (Imax)
Run / experiment:
Experiment 2
Value:
1.92
Vehicle controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, PF-06961030 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification