(Z)‐ethyl 2‐methylpent‐3‐enoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Nov 2020 - 02 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

18 June 2019

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 a weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

(Z)‐ethyl 2‐methylpent‐3‐enoate

EC Number:

854-058-4

Cas Number:

58625-89-1

Molecular formula:

C8H14O2

IUPAC Name:

(Z)‐ethyl 2‐methylpent‐3‐enoate

Test material form:

liquid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

TEST SYSTEM
The test system were 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.
Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Storage: The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months after 6 October 2020.

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions.
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.
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 RCcysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCIPA sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL IPA.
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.2 mg of SPCL in 19.69 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
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 RClysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCIPA sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL IPA.

- Preparation of the test chemical 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. Isopropanol (IPA) was selected as most appropriate solvent for test item stock solutions.

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.
For the cysteine and lysine reactivity assay respectively 23.69 mg and 27.44 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1666 μL and 1930 μL IPA, respectively, to obtain 100 mM solutions. Visual inspection of the formation 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.

- Positive control: Cinnamic aldehyde
Purity: 99.1%
Expiry of batch: 30 November 2021

- Preparation of the positive controls, and co-elution controls for SPCC
Three positive controls solutions (PCcys-1 to PCcys-3) were prepared by mixing 750 μL Stock solution of 0.667 mM SPCC with 200 μL ACN and 50 μL Cinnamic aldehyde solution (100 mM in ACN). One co-elution control (CClys-211886/A) was prepared by mixing 750 μL Ammonium acetate buffer pH 10.2 with 250 μL test item solution (100 mM).

- Preparation of the positive controls, and co-elution controls for SPCL
Three positive controls solutions (PClys-1 to PClys-3) were prepared by mixing 750 μL Stock solution of 0.667 mM SPCL with 200 μL ACN and 50 μL Cinnamic aldehyde solution (100 mM in ACN). One co-elution control (CClys-211886/A) was prepared by mixing 750 μL Ammonium acetate buffer pH 10.2 with 250 μL test item solution (100 mM).


INCUBATION
- Incubation conditions
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 times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 24.4 hours and 24.2 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
A SPCC and SPCL calibration curve was prepared with the following concentrations 0, 0.017, 0.033, 0.067, 0.133, 0.267, 0.534 mM.

- Verification of the suitability of the HPLC for test chemical and control substances
Analysis: All samples were analyzed according to the HPLC method presented in Table 1. (See 'other information on materials and methods').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2. (See 'other information on materials and methods').

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have a r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide peak areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.


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%
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 3), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Vehicle / solvent:

isopropanol

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 70.6% ±
0.7%.

The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.5% ±
0.7%.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Mean cysteine and lysine peptide depletion

Value:

2.1 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

1.4 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

2.8 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:

-Acceptance criteria of the calibration curve:
The correlation coefficient (r2) of the SPCC and SPCL standard calibration curve was 0.9995 and 1.0000, respectively. Since the r2 was >0.99, the calibration curves were accepted.

- Acceptance criteria met for positive control:
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 70.6% ± 0.7%. 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 60.5% ± 0.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

- Acceptance criteria met for reference controls A to C:
The mean SPCC peptide concentration of Reference Controls A was 0.535 ± 0.001 mM, the mean peptide concentration of Reference Controls C was 0.530 ± 0.004 mM and the mean peptide concentration of Reference Controls CIPA was 0.524 ± 0.002 mM. The means of Reference Control samples A, C and CIPA were all within the acceptance criteria of 0.50 ± 0.05 mM. The mean SPCL peptide concentration of Reference Controls A was 0.499 ± 0.003 mM, the mean peptide concentration of Reference Controls C was 0.504 ± 0.004 mM and the mean peptide concentration of Reference Controls CIPA was 0.510 ± 0.002 mM. The means of Reference Control samples A, C and CIPA 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 (IPA) used to dissolve the test item did not impact the Percent SPCC and SPCL Depletion.

- Acceptance criteria met for co-elution controls (Lysine and Cysteine):
The mean area ratio (A220/A258) of the SPCC Reference Control samples was 37.39. The mean A220/A258 ratio ± 10% range was 33.65-41.13. Each sample showed an A220/A258 ratio within these ranges. The mean area ratio (A220/A258) of the SPCL Reference Control samples was 30.03. The mean A220/A258 ratio ± 10% range was 27.03-33.03. Each sample showed an A220/A258 ratio within these ranges.

- Acceptance criteria met for variability between replicate measurements:
The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.3% and 1.2% for SPCC and SPCL, respectively. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

- See Table 4 in "any other information on results incl. tables" for an overview of the acceptability criteria.

Any other information on results incl. tables

- Solubility Assessment of the Test Item

The test item was soluble at a 100 mM concentration in multiple solvents, however, for almost all solvents precipitation of the test item after dilution in the SPCL assay buffer was observed except for IPA for which no solubility issues were observed. Based on these observations, IPA was selected as solvent for the main study.

- Results for the test item

Upon preparation as well as after incubation of the SPCC and SPCL with test item no precipitate or phase separation was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCC or SPCL. The mean SPCC A220/A258 area ratio was 37.24 (the mean was based on two values as the peak area at 258 nm for sample 211886/A-cys-3 was rejected, due to baseline disturbance). This was within the 33.65-41.13 range. The mean SPCL A220/A258 area ratio was 29.79. This was within the 27.04-33.03 range. The above demonstrates that there was no co-elution of the test item with SPCC or SPCL.

- DPRA predicition and reactivity classification

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 table 5. In the cysteine reactivity assay the test item showed 1.4% SPCC depletion while in the lysine reactivity assay the test item showed 2.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.1% and as a result the test item was 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.

Table 4. 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	0.9995	>0.99	1.0000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.535 ± 0.001	0.50 ± 0.05	0.499 ± 0.003
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.530 ± 0.004	0.50 ± 0.05	0.504 ± 0.004
Mean peptide concentration RC-CIPA samples (mM)	0.50 ± 0.05	0.524 ± 0.002	0.50 ± 0.05	0.510 ± 0.002
CV (%) for RC samples  B and C	<15.0	1.3	<15.0	1.2
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	70.6	40.2-69.0	60.5
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.7	<11.6	0.7
SD of peptide depletion for the test item (%)	<14.9	1.2	<11.6	0.8

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

Table 5. 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
Substance	1.4%	±1.2%	2.8%	±0.8%	2.1%	Negative: No or minimal reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Conclusions:

The substance was 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 (DPRA) study, performed according to OECD guideline 442C and in accordance with GLP principles, the reactivity of the Substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined. Isopropanol was found to be an appropriate solvent to dissolve the test substance in and was therefore used in this DPRA study. Cinnamic aldehyde was used as a positive control. Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (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. All acceptability criteria were met and therefore, the study was considered to be valid. No precipitate or phase separation was observed in any of the samples before and after the incubation. In the cysteine reactivity assay the test item showed 1.4% SPCC depletion and in the lysine reactivity assay the test item showed 2.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.1% 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.