Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 Feb 2019 - 13 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

A 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 one of the non-animal tests to be performed as part of weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Cas Number:

943986-70-7

Molecular formula:

C11H20N2O2

IUPAC Name:

Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Test material form:

solid

Details on test material:

- Appearance: Off-white solid
- Storage condition of test material: At room temperature protected from light

In chemico test system

Details on the study design:

TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. Acetonitrile (ACN) was evaluated and assessed to be suitable.
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 32.36 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1524 µL ACN after vortex mixing 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) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL (JPT Peptide Technologies GmbH, Berlin, Germany). The peptides were stored in the freezer (≤ -15°C) for a maximum of 6 months.

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. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.3 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC analysis the samples were visually inspected for precipitation. None of the samples showed precipitation.

Analysis: All samples were analyzed according to the HPLC method presented in the below Table (See 'other information on materials and methods').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in the other below Table (See 'other information on materials and methods').

DATA EVALUATION
The concentration of SPCC or SPCL was spectrophotometrically 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))]x100

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% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: Mean SPCC depletion (%)

Value:

0

Negative controls validity:

valid

Remarks:

CV between reference controls: 0.3%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 71.7 ± 0.1%

Remarks on result:

other: SD: 0.0%

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: Mean SPCL depletion (%)

Value:

0

Negative controls validity:

valid

Remarks:

CV between reference controls: 0.5%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 63.1 ± 1.3%

Remarks on result:

other: SD: 0.0%

Other effects / acceptance of results:

- Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
- No co-elution occured during both assays.

Any other information on results incl. tables

Acceptability criteria (which were all met):

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9999	>0.99	0.9998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.504 ± 0.001	0.50 ± 0.05	0.497 ± 0.002
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.504 ± 0.002	0.50 ± 0.05	0.497 ± 0.002
CV (%) for RC samples B and C	<15.0	0.3	<15.0	0.5
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	71.7	40.2-69.0	63.1
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.1	<11.6	1.3
SD of peptide depletion for the test item (%)	<14.9	0.0	<11.6	0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

other: Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

The substance was tested negative in the DPRA and was classified in the "No or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico study, performed according to OECD guideline 442C and GLP principles, the reactivity of the substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the substance to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers. Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by HPLC with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model. Acetonitrile was found to be an appropriate solvent to dissolve the test substance. Cinnamic aldehyde was used as a positive control. All acceptability criteria were met and therefore, the study was considered to be valid. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. No co-elution of the test item with SPCC or SPCL was observed. In the cysteine reactivity assay, as well as in the lysine reactivity assay, the test item showed 0.0% depletion. The mean of the SPCC and SPCL depletion was therefore also 0.0% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.