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Administrative data

Description of key information

DEREK assessment: negative

DPRA assay: negative

KeratinoSensTM assay: negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
in silico assessment
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
December 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
According to ECHA guidance R7a, available information, QSARs and in chemico/in vitro testing should be considered in a weight of evidence approach before in vivo testing as a last resort. The in silico DEREK assessment, the in chemico DPRA and the in vitro KeratinoSens assay are considered to give a reliable result for prediction of skin sensitization as the DEREK covers the whole process in the in silico assessment (including obvious metabolism) and the first steps of skin sensitization are covered in the DPRA and KeratinoSens assays.
Principles of method if other than guideline:
A DEREK assessment was performed.
GLP compliance:
no
Justification for non-LLNA method:
The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.
Since 11 October 2016 it is legally required to consider all available information for the endpoint skin sensitisation and to use a non in vivo test strategy based on in chemico, in silico and in vitro skin tests combined with a WoE. An in vivo test (LLNA) is only allowed as last resort.
Specific details on test material used for the study:
SMILES: COCC(NC(C)=O)C(=O)NCC1=CC=CC=C1
Key result
Parameter:
other: prediction skin sensitisation
Remarks on result:
no indication of skin sensitisation
Remarks:
DEREK predicted the substance to be not skin sensitising
Interpretation of results:
GHS criteria not met
Conclusions:
DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitisation for the test item SPM20200 is predicted to be not sensitising to the skin.
Executive summary:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitisation for the test item SPM20200 is predicted to be not sensitising to the skin.

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.
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
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

Details on 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%
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
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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the in vitro tests to be performed as part of weight of evidence.
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
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
pH (1% in water, indicative range): 7.3-7.0 (determined by Charles River Den Bosch)
Details on study design:
Preparation of test item solutions: No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM. The compound formed a clear colourless solution at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM. The stock and spike solution were diluted 25-fold with exposure medium resulting in concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.813 and 3.906 µM (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 µM (final concentration DMSO of 1%). All formulations formed a clear solution. All concentrations of the test item were tested in triplicate. Two independent runs were performed.

Positive control: ethylene dimethacrylate glycol (0.7.81 to 250 uM)

Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing, cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with a lid and then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium iwas removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second). The luminescence was measured with the TECAN Infinite® M200 Pro Plate Reader.

Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis: according to guideline
Interpretation of results: A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM should be considered as inconclusive.

Acceptance criteria:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells. If the variability is higher, results should be discarded.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.51 and the EC1.5 79.3 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.60 and the EC1.5 68.7 µM. At 15.6 µM almost no luminescence signal was measured. Probably no cells were added to these wells. This had no effect on the results since the other concentrations showed the expected results.
Key result
Run / experiment:
experiment 1
Remarks on result:
other: Imax 1.27-fold
Key result
Run / experiment:
experiment 2
Remarks on result:
other: Imax 1.40-fold
Other effects / acceptance of results:
Both experiments passed the acceptance criteria:
•The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
•The EC1.5 of the positive control was between 5 and 125 µM (79.3 µM and 68.7 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.51-fold and 2.60-fold in experiment 1 and 2, respectively).
•Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.7% and 9.5% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

SPM20200 showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.
Experiment 1: No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with SPM20200. The Imax was 1.27 and therefore no EC1.5 could be calculated.
Experiment 2: No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations. The Imax was 1.40 and therefore no EC1.5 could be calculated. One measurement at 125 µM was a clear outlier and was therefore excluded from the calculations.
Interpretation of results:
GHS criteria not met
Conclusions:
The KeratinoSensTM assay is valid and SPM20200 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

A KerationSensTM assay was performed with SPM20200 according to OECD 442D and GLP. SPM20200 was dissolved in dimethyl sulfoxide at 200 mM.From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977 – 2000 µM (2-fold dilution series). Two independent experiments were performed.

The test conditions were adequate and the test system functioned properly.

SPM20200 showed no toxicity (no IC30and IC50value) and no biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.27-fold and 1.40-fold in experiment 1 and 2 respectively. SPM20200 is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control.

It is concluded that the KeratinoSensTMassay is valid and that SPM20200 is classified as negative(no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes)under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For the DPRA assay SPM20200 dissolved sufficiently in acetonitrile, i.e. at 100 mM by visual inspection the solution was not cloudy and no precipitate was seen. For the KeratinoSensTM assay SPM20200 dissolved in DMSO at a concentration of 200 mM forming a clear colourless solution. As the test item could be solubilized, the STEP 1 studies were performed and the results are presented here.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item, and SPM20200 is predicted to be not sensitizing to the skin.

A valid DPRA test was performed according to OECD 442C and GLP. 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 reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model and was considered to be negative in the DPRA.

A valid KeratinoSensTM assay was performed according to OECD 442D and GLP. The maximum luciferase activity induction (Imax) was 1.27-fold and 1.40-fold in experiment 1 and 2, respectively. SPM20200 is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control.

The DEREK NEXUS assessment, the DPRA assay and the KeratinoSensTM assay were all negative for skin sensitization of SPM20200.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

based on the results from the DEREK assessment, the DPRA assay and the KeratinoSensTMassay, SPM20200 is not a skin sensitizer. Respiratory sensitisation is consequently also considered to be unlikely. SPM20200 does not need to be classified according to CLP Regulation 1272/2008 and amendments.