Propan-2-yl-oxocyclobutane carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05-11-2018 to 13-11-2018

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)

Test material

Test material information

1

Reference substance name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

EC Number:

834-970-9

Cas Number:

130111-95-4

Molecular formula:

C8H12O3

IUPAC Name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

Test material form:

liquid

Details on test material:

Batch (Lot) Number: GR13224
Expiry date: 31 January 2020 (expiry date)
Physical Description: Colourless to pale yellow liquid
Purity/Composition: 99.6%
Storage Conditions: At room temperature

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 71.5% ± 0.5%. 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 66.1% ± 1.5%. 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

Other effects / acceptance of results:

The correlation coefficient (r2) of the SPCC standard calibration curve was 1.000. Since the r
2 was >0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.514 ± 0.002 mM while the
mean peptide concentration of Reference Controls C was 0.512 ± 0.003 mM. The means of
Reference Control samples A and C were both within the acceptance criteria of
0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the
solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3).
The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and
C was 0.5%. 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 37.39.
The mean A220/A258 ratio ± 10% range was 33.65-41.13. 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 0.9999. Since the r2
was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.491 ± 0.011 mM, while the mean peptide concentration of Reference Controls C was
0.503 ± 0.003 mM. The means of Reference Control samples A, and C were both within the
acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and
indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent
SPCL Depletion.
The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3).
The CV of the peptide areas for the nine Reference Controls B and C was 0.4%. 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 31.33.
The mean A220/A258 ratio ± 10% range was 28.20-34.46. 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

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM  PF-06238566 stock solution in ACN 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. No precipitate or phase

separation was observed in any of the samples.

In 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 209802/A-cys samples, the mean SPCC A220/A258 area ratio was

37.37. Since this was within the 33.65-41.13 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 C. The mean Percent SPCC Depletion for the test item was 0.2% ± 0.1%.

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM  PF-06238566 stock solution in ACN 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. No precipitate or phase separation was observed in any

of the samples.

After incubation a precipitate was observed in the cinnamic aldehyde positive control

samples. As the mean Percent SPCL Depletion for the positive control cinnamic aldehyde

was within the acceptance range, it was evaluated as having no impact on the study results.

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 209802/A-lys  samples, the mean SPCL A220/A258

area ratio was 31.43. Since this was within the 28.20-34.46 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 C. The mean Percent SPCL Depletion for the Test Item was 1.9% ± 1.1%.

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 0.2% SPCC depletion while in the lysine reactivity assay

the test item showed 1.9% SPCL depletion. The mean of the SPCC and SPCL depletion was

1.0% 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.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

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-06238566 0.2% ±0.1% 1.9% ±1.1% 1.0%                             Negative: No or minimal reactivity

Applicant's summary and conclusion

Interpretation of results:

other: result used in Weight of Evidence assessment of AOP testing strategy

Conclusions:

In conclusion, this DPRA test is valid. PF-06238566 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.