isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14-01-2019 to 17-01-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In chemico study conducted as part of Adverse Outcome Pathway testing strategy

Test material

Test material information

Constituent 1

Reference substance name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

EC Number:

950-484-0

Molecular formula:

C9H17NO2*C4H6O4

IUPAC Name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13335
Expiry date: 31 January 2020 (expiry date)
Physical Description: White solid
Purity/Composition: 97.1%
Storage Conditions: At room temperature

In chemico test system

Details on the study design:

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.
Rationale Recommended test system in the international OECDguideline for DPRA studies.
Source JPT Peptide Technologies GmbH, Berlin, Germany.
Storage The peptides were stored in the freezer (≤-15°C) for a
maximum of 6 months.

Results and discussion

Positive control results:

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference
Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde
was 77.4% ± 0.4%. This was within the acceptance range of 60.8% to 100% with a SD that
was below the maximum (SD <14.9%).
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference
Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde
was 48.7% ± 2.1%. 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

Run / experiment:

other: 1

Parameter:

other: mean % SPCC depletion

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Run / experiment:

other: 1

Parameter:

other: mean % SPCL depletion

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9995. Since the r2 was >0.99,
the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was
0.514 ± 0.001 mM, the mean peptide concentration of Reference Controls C was
0.509 ± 0.002 mM and the mean peptide concentration of Reference Controls CMQ
was 0.503 ± 0.002 mM. The means of Reference Control samples A, C and CMQ
were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system
and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCC Depletion.
The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and
C was 1.1%. This was within the acceptance criteria (CV <15.0%) and confirms the stability
of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 37.66.
The mean A220/A258 ratio ± 10% range was 33.89-41.42. Each sample showing an A220/A258
ratio within this range gives an indication that co-elution has not occurred.

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9996. Since the r2 was >0.99,
the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was
0.507 ± 0.004 mM, the mean peptide concentration of Reference Controls C was
0.510 ± 0.003 mM and the mean peptide concentration of Reference Controls CMQ
was 0.503 ± 0.008 mM. The means of Reference Control samples A, C and CMQ
were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system
and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent
SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 0.9%. This was
within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over
time. The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12
(Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 31.74.
The mean A220/A258 ratio ± 10% range was 28.57-34.91. Each sample showing an
A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Any other information on results incl. tables

Data Evaluation

The concentration of SPCC or SPCL was spectrophotometrically determined at 220  nm  in

each sample by measuring the peak area of the appropriate peaks by peak integration and by

calculating the concentration of peptide using the linear calibration curve derived from the

standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area

and dividing it by the mean peak area of the relevant reference controls C according to the

following formula:

Percent Peptide Depletion = [1 - (Peptide Peak Area in Replicate Injection (at 220  nm) /Mean Peptide Peak Area in Reference Controls (at 220  nm))] x100

In addition, the absorbance at 258  nm  was determined in each sample by measuring the peak

area of the appropriate peaks by peak integration. The ratio of the 220  nm  peak area and the

258  nm  peak was used as an indicator of co-elution. For each sample, a ratio in the range of

90%<mean area ratio of control samples<110% gives a good indication that co-elution has

not occurred.

Data Interpretation

The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for

the test item. Negative depletion was considered as “0” when calculating the mean. By using

the Cysteine 1:10 / Lysine 1:50 prediction model (see table below), the threshold of 6.38%

average peptide depletion was used to support the discrimination between a skin sensitizer

and a non-sensitizer.

Mean of cysteine and lysine % depletion       Reactivity class              DPRA prediction

0% ≤ Mean % depletion ≤ 6.38%             No or minimal reactivity       Negative

6.38% < Mean % depletion ≤ 22.62%       Low reactivity                      Positive

22.62% < Mean % depletion ≤ 42.47%     Moderate reactivity               Positive

42.47% < Mean % depletion ≤ 100%        High reactivity                      Positive

Solubility Assessment of the Test Item

At a concentration of 100 mM,  PF-07097547-24 was not soluble in ACN, but was soluble in

MQ.  Therefore this solvent was used to dissolve the test item in this DPRA study.

Cysteine Reactivity Assay

The reactivity of  PF-07097547-24 towards SPCC was determined by quantification of the

remaining concentration of SPCC using HPLC analysis, following 23 hours of incubation at

25±2.5°C. Representative chromatograms of CCcys-209805/A and 209805/A-cys samples

are presented in Appendix 4. An overview of the retention time at 220  nm  and peak areas at

220  nm  and 258  nm  are presented in Table 3 (Appendix 3).

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM  PF-07097547-24 stock solution in  MQ  showed that the test item

was dissolved completely. Upon preparation and after incubation, both the co-elution control

(CC) as well as the test item samples were visually inspected.

The results of the cysteine reactivity assay for the test item are presented in Table 8

(Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see

chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test

item with SPCC.

For the 209805/A-cys samples, the mean SPCC A220/A258 area ratio was 37.49. Since this

was within the 33.89-41.42 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 CMQ. The mean Percent SPCC Depletion for the test item was 0.5% ± 0.5%.

Lysine Reactivity Assay

The reactivity of  PF-07097547-24 towards SPCL was determined by quantification of the

remaining concentration of SPCL using HPLC analysis, following 23 hours of incubation at

25±2.5°C. Representative chromatograms of CClys-209805/A and 209805/A-lys  samples are

presented in Appendix 4. An overview of the retention time at 220  nm  and peak areas at

220  nm  and 258  nm  are presented in Table 9 (Appendix 3).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM  PF-07097547-24 stock solution in  MQ  showed that the test item

was dissolved completely. Upon preparation and after incubation, both the CC as well as the

test item samples were visually inspected.

The results of the lysine reactivity assay for the test item are presented in Table 14

(Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see

chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test

item with SPCL.

For the 209805/A-lys  samples, the mean SPCL A220/A258 area ratio was 31.03. Since this was within the 28.57-34.91 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 CMQ. The mean Percent SPCL Depletion for the Test Item was 2.2% ± 1.3%.

DPRA Prediction and Reactivity Classification

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 0.5% SPCC depletion while in the lysine reactivity assay

the test item showed 2.2% SPCL depletion. The mean of the SPCC and SPCL depletion was

1.4% 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.

Applicant's summary and conclusion

Interpretation of results:

other: To be used in a weight of evidence approach for Skin sensitisation

Conclusions:

In conclusion, this DPRA test is valid. PF-07097547-24 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-07097547-24 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.

Milli-Q water  (MQ)  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 CMQ

, 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.

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 0.5% SPCC depletion while in the lysine reactivity assay

the test item showed 2.2% SPCL depletion. The mean of the SPCC and SPCL depletion was

1.4% 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.

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

PF-07097547-24 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.