Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 May 2020 - 18 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

EC Number:

950-492-4

Cas Number:

2361378-62-1

Molecular formula:

C21-23H39-43NO3

IUPAC Name:

Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

impurity 1

Reference substance name:

Amides, C16-18, N-(hydroxyethyl)

EC Number:

309-819-0

EC Name:

Amides, C16-18, N-(hydroxyethyl)

Cas Number:

101226-97-5

IUPAC Name:

Amides, C16-18, N-(hydroxyethyl)

impurity 2

Reference substance name:

Zirconium compounds

IUPAC Name:

Zirconium compounds

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D, OECD 442E and OECD 429 were also performed.

Background
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Solvent Selection
Based on the proportions of the main components of the test item together with their individual molecular weights, an apparent molecular weight of 373 g/mol was provided by the Sponsor and was used for preparation of the test item stock solutions.

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. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH). The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer based incubation mixtures. For the SPCC assay, a 20-fold dilution was prepared by mixing one volume of the test item stock solution with fifteen volumes of phosphate buffer pH 7.5 and four volumes of ACN. For the SPCL assay, a 4-fold dilution was prepared by mixing one volume of the test item stock solution with three volumes of ammonium acetate buffer pH 10.2. The presence of cloudiness, precipitate and/or phase separation was evaluated by visual inspection to aid solvent selection for the main study.
Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 62.62 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1679 μL EtOH after vortex mixing and 10 minute of sonication to obtain a 100 mM solution. Visual inspection of the clear solution being formed 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. Any residual volumes were discarded.

Controls
Reference controls, co-elution controls, positive control and negative controls were set up in parallel to the test item in order to confirm the validity of the test.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN. In addition, a RCcysCEtOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCEtOH sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL EtOH.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCEtOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCEtOH sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL EtOH.

Co-elution control
The co-elution control (CC) samples were:
Cysteine:750 μL Phosphate buffer pH 7.5,200 μL ACN, 50 μL test item/A test solution (100 mM)
Lysine:750 μL Ammonium acetate buffer pH 10.2, 250 μL test item/A test solution (100 mM)

Positive control
The positive controls were:
Cysteine:750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL Cinnamic aldehyde solution, (100 mM in ACN)
Lysine:750 μL Stock solution of 0.667 mM SPCL, 250 μL Cinnamic aldehyde solution (100 mM in ACN)

Peptides
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.0 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

Dose Groups
1. Co-elution control (CC)
2. Cinnamic aldehyde (PC) (100mM)
3. Test item
Cysteine: 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL test item/A test solution (100 mM)
Lysine:750 μL Stock solution of 0.667 mM SPCL, 250 μL 211324/A test solution (100 mM)

Experimental Procedure

Preparation of the HPLC Standard Calibration Curve: SPCC and SPCL Calibration Curves were prepared with 7 solutions from 0-0.534mM.

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 did not exceed 30 hours. Prior to HPLC analysis the samples were visually inspected for precipitation. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Preparation and Analysis

SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
System 2 (used for Lysine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
All samples were analyzed according to the HPLC method presented in Table 1 (Appendix 1).

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))] × 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

Positive control results:

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.3% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 63.0% ± 0.8%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: other: Test item

Parameter:

other: mean % cysteine depletion

Value:

20.7 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

72.3%

Results 2

Run / experiment:

other: other: Test item

Parameter:

other: mean % lysine depletion

Value:

15.6 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

63%

Results 3

Run / experiment:

other: other: Test item

Parameter:

other: Mean of % cysteine and lysine depletion

Value:

18.2 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Remarks:

low reactivity class

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature. Supernatants were transferred to new vials and analyzed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS: Refer to above

Any other information on results incl. tables

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted. The results of the Reference Control samples A, C and CEtOH are presented in Table 4 (Appendix 3). The means of Reference Control samples A, C and CEtOH were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (EtOH) used to dissolve the test item did not impact the Percent SPCC Depletion. The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 211324/A-cys samples, the mean SPCC A220/A258 area ratio was 37.02. Since this was within the 33.67-41.15 range, this again indicated that there was no co-elution of the test item with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls CEtOH. The mean Percent SPCC Depletion for the test item was 20.7% ± 0.9%.

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlation coefficient (r2) of the SPCL standard calibration curve was 1.000. Since the r2 was >0.99, the SPCL standard calibration curve was accepted. The results of the Reference Control samples A , C and CEtOH are presented in Table 10 (Appendix 3). The means of Reference Control samples A, C and CEtOH were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (EtOH) used to dissolve the test item did not impact the Percent SPCL Depletion.   The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at 220 nm at the retention time of SPCL (see chromatogram in Appendix 4). A small response was observed at 258 nm at the retention time of SPCL but this response was evaluated as having no impact on the study outcome. For the 211324/A-lys samples, the mean SPCL A220/A258 area ratio was 28.87. Since this was within the 27.45-33.55 range, this indicated that there was no co-elution of the test item with SPCL. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls CEtOH. The mean Percent SPCL Depletion for the test item was 15.6% ± 0.8%.

An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	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	0.998	>0.99	1
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.506 ± 0.004	0.50 ± 0.05	0.498 ± 0.006
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.490 ± 0.004	0.50 ± 0.05	0.478 ± 0.004
Mean peptide concentration RC-CEtOHsamples (mM)	0.50 ± 0.05	0.482 ± 0.007	0.50 ± 0.05	0.479 ± 0.006
CV (%) for RC  B and C samples	<15.0	1.3	<15.0	2.9
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	72.3	40.2-69.0	63
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1	<11.6	0.8
SD of peptide depletion for the test item (%)	<14.9	0.9	<11.6	0.8

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table belo.w

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Reaction mass of 2-(palmitoylamino)ethyl acrylate and 2-(stearoylamino) ethyl acrylate	20.70%	±0.9%	15.60%	±0.8%	18.20%	Positive: Low reactivity

Applicant's summary and conclusion

Interpretation of results:

other: WoE classification with OECD 442D, OECD 442E and OECD 429 studies.

Conclusions:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D, OECD 442E and OECD 429 were also performed. Under the experimental conditions of this study, the DPRA prediction is considered as positive and the test item was considered to have low reactivity for both peptides.

Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; 20243911), the test item (92.4%) in ethanol was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 ± 2 h at 25 ± 2.5 °C). Subsequently samples were analysed by HPLC. Reference controls (A, B, C (solvent control)), co-elution controls and a positive control (Cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and CEtOH, 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 all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature. Supernatants were transferred to new vials and analyzed. In the cysteine reactivity assay the test item showed 20.7% SPCC depletion while in the lysine reactivity assay the test item showed 15.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 18.2% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D, OECD 442E and OECD 429 were also performed. The data generated with this test will be considered in the context of an integrated approache such as IATA, combining the result with other complementary information from the other 3 tests.