methyl 2-hydroxybut-3-enoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18, corrected 2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
Cysteine peptide stock solution, 0.667 mM, 0.501 mg/mL: 0.01121 g of the peptide was pre-weighted and 20.158 mL of pH 7.5 phosphate buffer was added right before beginning the assay.
Lysine peptide stock solution, 0.667 mM, 0.518 mg/mL: 0.01033 g of the peptide was pre-weightedand 18.725 mL of pH 10.2 acetate buffer was added right before beginning of the assay.

- Preparation of the test chemical solutions: 100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.0234 g) based on the molecular weight and purity of the substance. 0.0234 g test chemical was weighted for the stock solutions used for the cysteine peptide depletion determination respectively and 0.0233 g test chemical was weighted for the stock solution used for lysine peptide depletion determination.

- Preparation of the positive controls, reference controls and co-elution controls
Positive control: 100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. The needed amount of test chemical was calculated (0.0664 g ± 10%) based on the molecular weight and purity of the substance. 0.0663 g cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0661 g cinnamaldehyde was weighted for the stock solution used for lysine peptide depletion determination in the runs.
Reference control A: Peptide stock solutions are combined with acetonitrile. System suitability is checked by the use of the three replicates of reference control A.
Reference control B: Peptide stock solutions are combined with acetonitrile. Stability of the peptides are checked by the use of the three replicates of reference control B, measured before and after of the reaction samples.
Reference control C: Peptide stock solutions are combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide). Three replicates of reference control C are used as a solvent control to which the peptide concentration/depletion of the reaction samples is compared. Since acetonitrile is not the chosen solvent for the test item, a reference control C with acetonitrile is prepared additionally as the solvent control for the positive control.
Co-elution controls: Test item stock solution (and acetonitrile in case of cysteine peptide) is combined with the respective buffer solutions in each run. Co-elution controls are used to check for test item and peptide co-elution.

INCUBATION
- Incubation conditions: The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5°C - 30°C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution
- Precipitation noted: No precipitation was noted.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20% acetonitrile:buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.
- Verification of the suitability of the HPLC for test chemical and control substances: Prior to routine use of the method, the laboratory demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 200 nm

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 67.22 % ± 0.51 % and with lysine peptide depletion value of 52.07 % ± 1.33 %.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.88 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no relevant increase

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

1.24 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no relevant increase

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, the laboratory demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for reference controls A to C: Yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The DPRA prediction must be considered in the framework of an IATA for a final prediction.

Conclusions:

In the in chemico Direct Peptide Reactivity Assay (DPRA) according to OECD guideline 442C, the test item showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions.

Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA) in accordance with OECD 442C and GLP. For the test item and the positive control substance, in order to derive a prediction two independent tests were evaluated, one with cysteine and one with lysine peptides. The results of two valid runs is used for the classification of the test item. Peptide depletion by the positive control cinnamaldehyde was 67.22 % ± 0.51 % for cysteine peptides and 52.07 % ± 1.33 % for lysine peptides, fulfilling all acceptance criteria for the positive control.

 

The mean back-calculated peptide concentrations of the reference control A replicates were within the expected molarity concentration range for cysteine (0.49 mM) and lysine peptides (0.50 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.9 % and 0.2 % percentages for cysteine and lysine peptides, respectively. Moreover, the mean back-calculated peptide concentrations of the three reference controls C in the ultrapure water replicates were within the expected molarity concentration range for cysteine (0.47 mM) and lysine peptides (0.50 mM). For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 1.24 % ± 2.72 % while the percent lysine peptide depletion was 1.88 % ± 0.46 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 1.56 %, which is well below the threshold of 6.38 % for the applicable prediction model. The test item fells therefore into the no or minimal reactivity class and is evaluated not to be a skin sensitizer in this DPRA test.

 

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method according to OECD 442C.