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EC number: 628-079-2 | CAS number: 3680-69-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Nanomaterial surface chemistry
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 03-12-2018 to 06-12-2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- 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
- Reference substance name:
- 4-chloro-7H-pyrrolo[2,3-d]pyrimidine
- EC Number:
- 628-079-2
- Cas Number:
- 3680-69-1
- Molecular formula:
- C6H4ClN3
- IUPAC Name:
- 4-chloro-7H-pyrrolo[2,3-d]pyrimidine
- Test material form:
- solid
- Details on test material:
- Batch (Lot) Number: GR13290
Expiry date: 31 January 2020 (expiry date)
Physical Description: White to off-white solid
Purity/Composition: 99.1%
Storage Conditions: At room temperature
Constituent 1
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 74.2% ± 0.2%. 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 64.8% ± 1.1%. 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
Resultsopen allclose all
- Run / experiment:
- other: 1
- Parameter:
- other: mean % SPCC depletion
- Value:
- 3.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Run / experiment:
- other: 1
- Parameter:
- other: mean % SPCL depletion
- Value:
- 0.1
- 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 0.9994. Since the r2 was >0.99,
the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was
0.521 ± 0.003 mM, the mean peptide concentration of Reference Controls C was
0.527 ± 0.011 mM and the mean peptide concentration of Reference Controls CIPA
was 0.527 ± 0.012 mM. The means of Reference Control samples A, and C and CIPA
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 (IPA) 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 2.0%. This was within the acceptance criteria (CV <15.0%) and confirms the stability
of the HPLC run over time.
The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6
(Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 37.87.
The mean A220/A258 ratio ± 10% range was 34.08-41.66. 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
0.473 ± 0.009 mM, the mean peptide concentration of Reference Controls C was
0.481 ± 0.003 mM and the mean peptide concentration of Reference Controls CIPA
was 0.480 ± 0.005 mM. The means of Reference Control samples A, C and CIPAt
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 (IPA) 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 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 31.24.
The mean A220/A258 ratio ± 10% range was 28.12-34.37. 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-01323624 was not soluble in ACN, MQ, ACN:MQ (1:1,
v/v), but was soluble in IPA and acetone:ACN (1:1, v/v). IPA was used to dissolve the test
item in this DPRA study.
Cysteine Reactivity Assay
The reactivity of PF-01323624 towards SPCC was determined by quantification of the
remaining concentration of SPCC using HPLC analysis, following 23.8 hours of incubation at
25±2.5°C. Representative chromatograms of CCcys-209807/A and 209807/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-01323624 stock solution in IPA 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.
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 209807/A-cys samples, the mean SPCC A220
/A258area ratio was38.60. Since this was within the 34.08-41.66 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 CIPA. The mean Percent SPCC Depletion for the test item was 3.3% ± 1.3%.
Lysine Reactivity Assay
The reactivity of PF-01323624 towards SPCL was determined by quantification of the
remaining concentration of SPCL using HPLC analysis, following 23.8 hours of incubation at
25±2.5°C. Representative chromatograms of CClys-209807/A and 209807/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-01323624 stock solution in IPA 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 test item 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.
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 209807/A-lys samples, the mean SPCL A220
/A258 area ratio was 30.96. Since this was within the 28.12-34.37 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 CIPA. The mean Percent SPCL Depletion for the Test Item was 0.1% ± 0.2%.
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 3.3% 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
1.7% 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-01323624 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-01323624 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.
Isopropanol (IPA) 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 CIPA, 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 3.3% 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
1.7% 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.
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