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EC number: 210-498-3 | CAS number: 616-91-1
- 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
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-01-24 to 2018-03-10
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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:
- February 04, 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
- Version / remarks:
- January 12, 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- Acetylcysteine
- EC Number:
- 210-498-3
- EC Name:
- Acetylcysteine
- Cas Number:
- 616-91-1
- Molecular formula:
- C5H9NO3S
- IUPAC Name:
- N-acetylcysteine
Constituent 1
In chemico test system
- Details on the study design:
- The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Controls:
Positive control (PC) : Cinnamic aldehyde in acetonitrile (100 mM stock solution)
Sigma Aldrich; Cat. No.: W228613; ≥95%; CAS No.: 104-55-2; Lot. No.: MKBX1392V
Co-elution control: set up in parallel to sample preparation but without respective peptide solution
Reference controls (RCs): set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
HPLC System: see "Any other information on materials"
Test System
Peptides with > 95 % purity, synthesized by JPT Peptide Technologies GmbH, were used.
Cys-Peptide (Cysteine): Lot. No.: 111016HS_MHeW1017
Lys-Peptide (Lysine): Lot. No.: 120514HSDW_W1117
20.15 mg (experiment 1) and 19.65 mg (experiment 2) cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.18 mL: experiment 1; 38.10 mL: experiment 2) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
19.32 mg (experiment 1) and 20.42 mg (experiment 2) lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (36.69 mL: experiment 1; 38.78 mL: experiment 2) to reach a concentration of 0.667 mM
Solubility of test item
The test item was completely soluble in acetonitrile, therefore, acetonitrile was chosen as suitable vehicle for the main experiments.
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400xg) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.
Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for both, the cysteine and the lysine peptide.
Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM)
using the respective DB was performed, resulting in 7 calibration solutions covering the range 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017/ 0.00 mM peptide.
HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30°C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
Results and discussion
- Positive control results:
- The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.89% (experiment 1) and 69.46% (experiment 2). No co-elution of the test item with any of the peptide peaks was observed.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1st experiment
- Parameter:
- other: mean peptide depletion of both peptides [%]
- Value:
- 6.28
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2st experiment
- Parameter:
- other: peptide depletion of both peptides (%)
- Value:
- 2.16
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
All acceptance criteria are fulfiled
- 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: Depletion of the Cysteine Peptide (experiment 1)
Cysteine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1371.9458 |
0.1446 |
72.07 |
72.27 |
0.27 |
0.38 |
1367.3362 |
0.1441 |
72.17 |
||||
1346.7943 |
0.1419 |
72.58 |
||||
Test Item |
4922.7578 |
0.5198 |
0.00 |
0.00 |
0.00 |
- |
4953.7280 |
0.5231 |
0.00 |
||||
4937.0298 |
0.5214 |
0.00 |
Table 2: Depletion of the Cysteine Peptide (experiment 2)
Cysteine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
3.7100 |
0.1138 |
77.40 |
77.79 |
0.34 |
0.44 |
3.6200 |
0.1110 |
77.95 |
||||
3.6080 |
0.1106 |
78.02 |
||||
Test Item |
17.3900 |
0.5340 |
0.00 |
0.00 |
0.00 |
- |
16.7550 |
0.5145 |
0.00 |
||||
16.7130 |
0.5132 |
0.00 |
Table 3: Depletion of the Lysine Peptide (experiment 1)
Lysine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1489.3650 |
0.1777 |
64.50 |
63.51 |
0.86 |
1.35 |
1548.7262 |
0.1848 |
63.08 |
||||
1554.0549 |
0.1854 |
62.95 |
||||
Test Item |
3723.8833 |
0.4440 |
11.23 |
12.57 |
1.20 |
9.52 |
3652.0933 |
0.4354 |
12.94 |
||||
3627.1846 |
0.4324 |
13.54 |
Table 4: Depletion of the Lysine Peptide (experiment 2)
Lysine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1548.8514 |
0.1877 |
62.22 |
61.12 |
1.28 |
2.09 |
1581.2108 |
0.1916 |
61.43 |
||||
1651.4639 |
0.2002 |
59.72 |
||||
Test Item |
3939.2124 |
0.4789 |
3.91 |
4.32 |
0.76 |
17.62 |
3941.6335 |
0.4792 |
3.85 |
||||
3886.3860 |
0.4725 |
5.20 |
Table 5: Categorization of the Test Item (experiment 1)
Prediction Model |
Prediction Model 1 |
Prediction Model 2 |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
6.28 |
Minimal Reactivity |
no sensitiser |
0.00 |
Minimal Reactivity |
no sensitiser |
Positive Control |
67.89 |
High Reactivity |
sensitiser |
72.27 |
Moderate Reactivity |
sensitiser |
Table 6: Categorization of the Test Item (experiment 2)
Prediction Model |
Prediction Model 1 |
Prediction Model 2 |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
2.16 |
Minimal Reactivity |
no sensitiser |
0.00 |
Minimal Reactivity |
no sensitiser |
Positive Control |
69.46 |
High Reactivity |
sensitiser |
77.79 |
Moderate Reactivity |
sensitiser |
Applicant's summary and conclusion
- Interpretation of results:
- other: no peptide binding
- Conclusions:
- Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows minimal reactivity in the DPRA under the test conditions chosen.
- Executive summary:
A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item N-Acetyl-L-cysteine towards cysteine (Cys-) and lysine (Lys-) containing peptides. The test substance was dissolved in acetonitrile and a stock solution of 100 mM was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. All test item solutions were freshly prepared immediately prior to use.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide or lysine peptide solution in both independent experiments. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples in both independent experiments.
Phase separation was observed for the co-elution of the positive control in both independent experiments and for the positive control in the second experiment.
Since the acceptance for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity or phase separation were regarded as insignificant.
Experiment 1:
No co-elution of test item with the peptide peaks was observed. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (6.28%). Based on the prediction model 1 the test item can be considered as non-sensitiser. According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered for both peptides. Therefore, no prediction can be made.
Experiment 2:
No co-elution of test item with the peptide peaks was observed. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (2.16%).
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.89% (experiment 1) and 69.46% (experiment 2).
In this study under the given conditions the test item showed minimal reactivity towards both peptides.
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