N-((3R,6S)-6-methylpiperidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th February 2020 to 14th February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

1

Reference substance name:

Reference substance 001

Cas Number:

2374827-92-4

Test material form:

solid: particulate/powder

In chemico test system

Details on the study design:

Preparation of test item
A correction factor of 1.08 was used to correct for purity/composition of the test item. Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An apppropriate 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 (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol. Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 41.73 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 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.
Test System
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Sample Incubation
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 times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 24.6 hours and 24.7 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours. Prior to HPLC analysis the samples were visually inspected for precipitation.
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)

Results and discussion

Positive control results:

Lysine Reactivity Assay - The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 62.8% ± 0.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Cysteine Reactivity Assay - The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 70.5% ± 1.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%)

In vitro / in chemico

Results

Results 1

Run / experiment:

other: SPCL depletion

Parameter:

other: Cysteine 1:10 / Lysine 1:50

Value:

0.2

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Negative: No or minimal reactivity

Results 2

Run / experiment:

other: SPCC depletion

Parameter:

other: Cysteine 1:10 / Lysine 1:50

Value:

0.5

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no or minimal reactivity class

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.
PF-06715298 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.

Executive summary:

The objective of this study was to determine the reactivity of PF-06715298 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 spectrophotometric 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 nonsensitizers.
The study procedures described in this report were based on the most recent OECD guideline.
Methanol (MeOH) 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, C and CMeOH, 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, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed 3.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.1% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.6% 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.