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EC number: 603-290-2 | CAS number: 128625-52-5
- 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
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- Auto flammability
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- Stability: thermal, sunlight, metals
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- Endpoint summary
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- 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
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- 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:
- weight of evidence
- Study period:
- 24 June 2019 - 11 July 2019
- 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- 4 February 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EC method B.59: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), Commission Regulation (EU) No 2017/735
- Version / remarks:
- February 14, 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
- EC Number:
- 603-290-2
- Cas Number:
- 128625-52-5
- Molecular formula:
- C18H28N6OP2F6
- IUPAC Name:
- Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
Constituent 1
In chemico test system
- Details on the study design:
- Skin sensitisation (In chemico test system):
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C.
Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.668 mM in pH 10.2 ammonium acetate buffer
Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 156.06 mg test item was dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.
Positive control, reference controls and co-elution control
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a co-elution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible co-elution of the test item with either the lysine or the cysteine peptide.
Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analysed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis. If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.
Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.
Data evaluation
HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) started 22 to 26 hours after the test item had been mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours. The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
Acceptance criteria
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.
The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should 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 should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.
Prediction model:
The mean percent cysteine and percent lysine depletion value was calculated for each test item. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an Integrated Approach to Testing and Assessment (IATA). Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate and high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA.
Results and discussion
- Positive control results:
- Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.
In vitro / in chemico
Results
- Key result
- Run / experiment:
- run/experiment 1
- Parameter:
- other: mean peptide depletion
- Value:
- 8.85
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- OTHER EFFECTS:
- No precipitate in the reaction mixture at the end of the incubation time and no co-elution were observed.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Any other information on results incl. tables
TABLE 1 Mean of cy steine and lysine % depletion
Test item Art. 851009 |
|||||
Cysteine peptide |
|||||
Sample no. |
Peak area |
Actual |
Peak area |
Peptide |
Corrected |
Replicate 1 |
45.5235 |
0.471 |
49.0051 |
6.83 |
6.83 |
Mean |
45.661 |
0.473 |
48.8631 |
6.55 0.272 0.042 |
6.55 |
Lysine peptide |
|||||
Sample no. |
Peak area |
Actual |
Peak area |
Peptide |
Corrected [%] |
Replicate 1 |
35.2319 |
0.437 |
39.4984 |
10.82 |
10.82 |
Mean |
35.105 |
0.436 |
39.5050 0.0180 0.0005 |
11.14 |
11.14 0.285 |
Acceptance criteria: |
SD % depletion Cysteine < 14.9 met: |
YES |
|||
Result: |
Art. 851009 |
|
|
|
|
Mean |
Reactivity |
Reactivity |
SD =
standard deviation
CV = coefficient of variation
TABLE 1 Mean of cysteine and lysine % depletion (continued)
Positive control: Cinnamic aldehyde, vehicle: acetonitrile |
|||||
Cysteine peptide
|
|||||
Sample |
Peak area |
Actual |
Peak area |
Peptide |
Corrected |
no. |
|
peptide |
reference |
depletion |
peptide |
|
|
[mM] |
control C |
[%] |
depletion |
Replicate 1 |
14.9565 |
0.154 |
49.0051 |
69.39 |
69.39 |
Replicate 2 |
14.5266 |
0.150 |
48.9476 |
70.27 |
70.27 |
Replicate 3 |
14.7173 |
0.152 |
48.6365 |
69.88 |
69.88 |
Mean |
14.733 |
0.152 |
48.8631 |
69.85 |
69.85 |
SD |
0.215 |
0.002 |
0.1983 |
0.441 |
0.441 |
CV |
0.015 |
0.015 |
0.0041 |
0.006 |
0.006 |
Lysine peptide |
|||||
Sample |
Peak area |
Actual |
Peak area |
Peptide |
Corrected |
no. |
|
peptide |
reference |
depletion |
peptide |
|
|
[mM] |
control C |
[%] |
depletion |
Replicate 1 |
16.7054 |
0.206 |
39.4984 |
57.71 |
57.71 |
Replicate 2 |
17.1234 |
0.212 |
39.4912 |
56.66 |
56.66 |
Replicate 3 |
17.3847 |
0.215 |
39.5254 |
55.99 |
55.99 |
Mean |
17.071 |
0.211 |
39.5050 |
56.79 |
56.79 |
SD |
0.343 |
0.004 |
0.0180 |
0.867 |
0.867 |
CV |
0.020 |
0.020 |
0.0005 |
0.015 |
0.015 |
Acceptance criteria: |
60.8 < Mean % depletion Cysteine < 100 |
YES |
|||
|
|
40.2< Mean % depletion Lysine < 69.0 |
YES |
||
|
|
SD % depletion Cysteine < 14.9 |
|
YES |
|
|
|
SD % depletion |
Lysine < 11.6 |
|
YES |
SD =
standard deviation
CV = coefficient of variation
Applicant's summary and conclusion
- Interpretation of results:
- other: peptide depletion positive
- Conclusions:
- In this study under the given conditions the test item is considered positive regarding peptide depletion and predicted to be a sensitiser (low reactivity). The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).
- Executive summary:
A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.
Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.
Test item-treated samples revealed a cysteine peptide depletion of 6.55% and a lysine peptide depletion of 11.14% (mean peptide depletion of 8.85%) and, hence, were below 22.62% and above 6.38%. The test item is considered positive and predicted to be a sensitiser (low reactivity) in the Direct Peptide Reactivity Assay (DPRA).
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