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Skin sensitisation

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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:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
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 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.