Reaction mass of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide and tertbutyl methyl ether

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th August 2021 to 12th October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

Updated 442C (14 June 2021)

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; 2-methoxy-2-methylpropane

EC Number:

955-078-7

Cas Number:

2755812-44-1

Molecular formula:

C28H44F3N5O5

IUPAC Name:

(1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; 2-methoxy-2-methylpropane

Test material form:

solid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Preparation of Test Item
A correction factor of 1.12 was used to correct for purity/composition of the test item.
Concentrations are expressed as active ingredient.
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 (IPA) and acetone:ACN (1:1, v/v).
The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer based incubation mixtures. For the SPCC assay, a 20-fold dilution was prepared by mixing one volume of the test item stock solution with fifteen volumes of phosphate buffer pH 7.5 and four volumes of ACN. For the SPCL assay, a 4-fold dilution was prepared by mixing one volume of the test item stock solution with three volumes of ammonium acetate buffer pH 10.2. The presence of cloudiness, precipitate and/or phase separation was evaluated by visual inspection to aid solvent selection for the main study.
Test item stock solutions were prepared freshly for each reactivity assay.
For the cysteine and lysine reactivity assay respectively 117.7 mg and 115.7 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1788 uL and 1758 wL acetone: ACN (1:1, v/v), respectively, to obtain 100 mM solutions. 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.

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 24.4 hours and 24.5 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 absorbance detector 2487 (Waters)

System 2 (used for Lysine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual absorbance detector 2487 (Waters)

Vehicle / solvent:

1:1 mix, acetone:acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 70.1% + 0.1%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 65.8% + 0.3%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Parameter:

mean lysine depletion

Value:

0.4 %

At concentration:

0.048 mM

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Parameter:

mean cystein depletion

Value:

3.3 %

At concentration:

0.486 mM

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have a r7>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between A0.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 + 0.05 mM.
e) The Coefficient of Variation (CV) of peptide peak areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 + 0.05 mM.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this DPRA test is valid. PF-07321332 MBTE Solvate 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-07321332 MBTE Solvate 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 assigns 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 442C guideline.
Acetone:acetonitrile (ACN) (1:1, v/v) 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, 1.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and C acetone:acn, 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 as stated in the OECD 442C guideline.

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.
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 3.3% SPCC depletion while in the lysine reactivity assay the test item showed 0.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.0% 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.

 

Test Item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	+/- SD	Mean	+/- SD		Cysteine 1:10 / Lysine 1:50 prediction model
PF-07320267 MBTE Solvate	3.3%	+/- 0.5%	0.8%	+/- 0.4%	2.0%	Negative: No or minimal reactivity

In conclusion, as all acceptability criteria were met this DPRA is considered to be valid.
PF-07321332 MBTE Solvate 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.