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EC number: - | CAS number: -
- 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:
- key study
- Study period:
- 12th September 2016 - completed 18th January 2017 - reported 23rd February 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154,
January 12, 2013 - Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- ECHA Guidelines 7a July 2017, require the use of this test prior to the LLNA as animal testing is a last resort. The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP .
[4], [12]. It is therefore a crucial step for the sensitising potential of a chemical.
Test material
- Test material form:
- liquid
- Details on test material:
- Chemical Name: N-[2-(2-Hydroxyethoxy)ethyl]acetamide
CAS No.: 118974-46-2
Batch: 27191705
Purity: 82% (dose calculation was adjusted to puriy)
Appearance: Pale to yellow liquid
Expiry Date: 16 June 2017
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical
Constituent 1
In chemico test system
- Details on the study design:
- Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
Controls
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference Control
Reference controls (RCs) were 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 test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. 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.
Peptides
20.36 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.532 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
21.37 mg 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 (37.732 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
Dose Groups
Reference Control C (solvent control) undiluted
Test Item 100 mM stock solution
Positive Control 100 mM stock solution
Pre-Experiments
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). The test item was dissolved in the following solvents suitable for the test:
- acetonitrile
- water
The test item was soluble in water, which was used as solvent for the test.
Experimental Procedure
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. Samples were prepared according to the scheme described in Table 2.
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Key result
- Parameter:
- other: Peptide depletion %
- Remarks:
- Prediction model 1
- Value:
- 0.46
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Parameter:
- other: Preptide depletion %
- Remarks:
- Prediction Model 2
- Value:
- 0.59
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
Any other information on results incl. tables
Pre-Experiments
Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was soluble in water. No turbidity,
precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using water.
Precipitation and Phase Separation
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 solution.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples. A phase separation was observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.
Since the positive control fulfilled all quality criteria the observed precipitations were considered as irrelevant.
Co-elution with the Peptide Peaks
No co-elution of the test item with any of the peptide peaks was observed.
Table 6 Cysteine and Lysine Values of the Calibration Curve
Sample |
Cysteine Peptide |
Lysine Peptide |
||
Peak Area at 220 nm |
Peptide Concentration [mM] |
Peak Area at 220 nm |
Peptide Concentration [mM] |
|
STD1 |
4655.6279 |
0.5340 |
4493.0674 |
0.5340 |
STD2 |
2355.5945 |
0.2670 |
2277.1133 |
0.2670 |
STD3 |
1163.3136 |
0.1335 |
1160.3005 |
0.1335 |
STD4 |
583.4137 |
0.0667 |
577.1267 |
0.0667 |
STD5 |
288.7250 |
0.0334 |
292.0436 |
0.0334 |
STD6 |
140.0172 |
0.0167 |
147.2783 |
0.0167 |
STD7 |
0.0000 |
0.0000 |
0.0000 |
0.0000 |
Based on these results, linear regression was performed and the calibration curves were determined.
Results of the Cysteine Peptide Depletion
Table 7: Results of the Cysteine Peptide Depletion
Cysteine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1333.1064 |
0.1526 |
69.69 |
70.06 |
0.48 |
0.68 |
1323.4985 |
0.1515 |
69.91 |
||||
1292.8408 |
0.1479 |
70.60 |
||||
Test Item |
4333.3467 |
0.4960 |
0.00 |
0.59 |
0.64 |
109.09 |
4306.2905 |
0.4929 |
0.49 |
||||
4272.4668 |
0.4890 |
1.27 |
Table 8: Results of the Lysine Peptide Depletion
Lysine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1802.2493 |
0.2125 |
57.49 |
57.48 |
1.25 |
2.18 |
1749.5433 |
0.2062 |
58.73 |
||||
1855.6707 |
0.2188 |
56.23 |
||||
Test Item |
4122.5361 |
0.4883 |
0.18 |
0.33 |
0.14 |
43.44 |
4110.8330 |
0.4870 |
0.46 |
||||
4115.9360 |
0.4876 |
0.34 |
Table 9: Categorization of the Test Item
Based on the results of the peptide depletion, categorization according to the prediction model might be performed. Since precipitation of the lysine peptide was observed, prediction model 2 should be considered.
Predicition Model |
Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50) |
Prediction Model 2 (Cysteine Peptide / TestItemRatio: 1:10) |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
0.46 |
Minimal Reactivity |
no sensitizer |
0.59 |
Minimal Reactivity |
no sensitizer |
Positive Control |
63.77 |
High Reactivity |
sensitizer |
70.06 |
High Reactivity |
sensitizer |
Table 10 Acceptance Criteria for Cysteine Peptide
Cysteine Peptide Run |
|||
Acceptance Criterion |
Range |
Value |
pass/fail |
coefficient of determination |
R² > 0.99 |
1.0000 |
pass |
mean peptide concentration of RC A |
0.45≤x≤0.55 mM |
0.5149 |
pass |
mean peptide concentration of RC C (PC) |
0.45≤x≤0.55 mM |
0.5033 |
pass |
mean peptide concentration of RC C (TI) |
0.45≤x≤0.55 mM |
0.4953 |
pass |
CV of the peak area of RC B |
< 15% |
1.54 |
pass |
CV of the peak area of RC C (PC) |
< 15% |
0.77 |
pass |
CV of the peak area of RC C (TI) |
< 15% |
1.43 |
pass |
mean peptide depletion of the PC |
60.8% < x < 100% |
70.06 |
pass |
SD of peptide depletion of the PC replicates |
< 14.9% |
0.48 |
pass |
SD of peptide depletion of the TI replicates |
< 14.9% |
0.64 |
pass |
Table 11 Acceptance Criteria for Lysine Peptide
Lysine Peptide Run |
|||
Acceptance Criterion |
Range |
Value |
pass/fail |
coefficient of determination |
R² > 0.99 |
0.9999 |
pass |
mean peptide concentration of RC A |
0.45≤x≤0.55 mM |
0.5024 |
pass |
mean peptide concentration of RC C (PC) |
0.45≤x≤0.55 mM |
0.5023 |
pass |
mean peptide concentration of RC C (TI) |
0.45≤x≤0.55 mM |
0.4892 |
pass |
CV of the peak area of RC B |
< 15% |
0.28 |
pass |
CV of the peak area of RC C (PC) |
< 15% |
0.15 |
pass |
CV of the peak area of RC C (TI) |
< 15% |
0.18 |
pass |
mean peptide depletion of the PC |
40.2% < x < 69.0% |
57.48 |
pass |
SD of peptide depletion of the PC replicates |
< 11.6% |
1.25 |
pass |
SD of peptide depletion of the TI replicates |
< 11.6% |
0.14 |
pass |
Historical Data
Table 12 Historical Data Cysteine Peptide
Cysteine Peptide |
|||
|
mean |
SD |
N |
linearity of the calibration curve |
0.9995 |
0.0005 |
22 |
mean peptide concentration of reference A [mM] |
0.5019 |
0.0000 |
22 |
mean peptide concentration of reference C [mM] |
0.4873 |
0.0000 |
36 |
CV of the peak area of control B [%] |
2.43 |
1.39 |
22 |
CV of the peak area of control C [%] |
1.72 |
1.45 |
36 |
mean peptide depletion of the PC [%] |
73.11 |
1.54 |
22 |
SD of peptide depletion of the PC replicates [%] |
0.69 |
0.45 |
22 |
SD of peptide depletion of the test items [%] |
13.02 |
26.77 |
57 |
Table 13 Historical Data Lysine Peptide
Lysine Peptide |
|||
|
Mean |
SD |
N |
linearity of the calibration curve |
0.9999 |
0.0001 |
21 |
mean peptide concentration of reference A [mM] |
0.4868 |
0.0157 |
21 |
mean peptide concentration of reference C [mM] |
0.4868 |
0.1938 |
34 |
CV of the peak area of control B [%] |
0.64 |
0.19 |
21 |
CV of the peak area of control C [%] |
0.50 |
0.62 |
34 |
mean peptide depletion of the PC [%] |
59.28 |
6.86 |
21 |
SD of peptide depletion of the PC replicates [%] |
1.67 |
1.48 |
21 |
SD of peptide depletion of the test items [%] |
1.62 |
2.33 |
57 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions the test item showed minimal reactivity towards the cysteine and lysine peptide. The test item might be considered as “non-sensitizer”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach
such as IATA. - Executive summary:
Results
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.
In the present study N-[2-(2-Hydroxyethoxy)ethyl]acetamide was given into water, based on the results of the pre-experiments. Based on the weighted molecular weight of 137.26 g/mol a 100 mM
stock solution 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.
The test item was completely soluble and the resulting solution was used for further testing.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation.
For the cysteine peptide experiment no precipitation, turbidity or phase separation was observed for any test sample.
For the lysine peptide experiment no precipitation, turbidity or phase separation was observed for the test item samples. A phase separation was observed for the samples of the positive control and
the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.
No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by
comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).
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% (0.46%). Based on prediction model 1 the test
item can be considered as non-sensitiser.
Since the positive control fulfilled all quality criteria the observed precipitations were considered as irrelevant. 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 63.77%.
Conclusion
In this study under the given conditions the test item showed minimal reactivity towards the cysteine and the lysine peptide. The test item might be considered as “non-sensitizer”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach
such as IATA.
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