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EC number: 700-539-8 | CAS number: 175481-26-2
- 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:
- Nov - Dec 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.
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:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- N-benzyl-2-acetamido-3-methoxypropanamide
- EC Number:
- 700-539-8
- Cas Number:
- 175481-26-2
- Molecular formula:
- C13H18N2O3
- IUPAC Name:
- N-benzyl-2-acetamido-3-methoxypropanamide
- Test material form:
- solid: particulate/powder
- Details on test material:
- - Appearance: White powder
Constituent 1
- Specific details on test material used for the study:
- Chemical name (IUPAC), synonym or trade name: 2-acetamido-N-benzyl-3-methoxy-propanamide
CAS Number: 175481-26-2
Molecular formula C13H18N2O3
Molecular weight 250.3 g/mol
In chemico test system
- Details on the study design:
- Skin sensitisation (In chemico test system) - Details on study design:
Test item preparation: No correction was made for the purity/composition of the test item.
Solubility of the test item was assessed before performing the DPRA assay. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
Test item stock solutions were prepared freshly for each reactivity assay.
For the cysteine reactivity assay 18.61 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 790 µL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) reactivity assay.
For the lysine reactivity assay 30.55 mg of the test item was pre-weighed into a clean amber vial and dissolved, just before use, in 1221 µL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the lysine (lys) reactivity assays.
Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL.
Source: JPT Peptide Technologies GmbH, Berlin, Germany
Rationale: recommended test system in the international OECD guideline for DPRA studies.
Calibration curve SPCC and SPCL: according to guideline
Positive control: cinnamic aldehyde
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler, in the dark, and incubated at 25±2.5°C for 24±2 hours.
Prior to HPLC-PDA analysis the samples were visually inspected. None of the samples showed precipitation.
Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 20 (APPENDIX 3). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 21 (APPENDIX 4).
The time between the first injection (= RCcysB-1 or RClysB-1) and the last injection of the sequence did not exceed 30 hours.
Data evaluation: The concentration of SPCC or SPCL was photometrically 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)))]×100
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%
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Key result
- Parameter:
- other: SPCC
- Remarks:
- mean percentage
- Value:
- 5.6 %
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Parameter:
- other: SPCL
- Remarks:
- mean percentage
- Value:
- 0.4 %
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- It can be concluded that this DPRA test is valid, and that SPM20200 was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
- Executive summary:
In this study the reactivity of SPM20200 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined according to OECD 442C and GLP to categorize SPM20200 in one of four classes of reactivity to support the discrimination between skin sensitisers and nonsensitisers. Following 24 hours of 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 photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated. Acetonitrile was used to dissolve the test item.
The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, 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 within the acceptability criteria for the DPRA assay.No co-elution of the test item with SPPC or SPCL was observed.
In the cysteine reactivity assay the test item showed 5.6% SPCC depletion, and in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 3.0%. As a result, SPM20200 was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model and was considered to be negative in the DPRA.
It can be concluded that this DPRA test is valid, and that SPM20200 was negative in the DPRA and is classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
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