Methyl 2,3-dibromopropionate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-02-05 to 2019-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

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:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2,3-dibromopropionate

EC Number:

217-044-3

EC Name:

Methyl 2,3-dibromopropionate

Cas Number:

1729-67-5

Molecular formula:

C4H6Br2O2

IUPAC Name:

methyl 2,3-dibromopropanoate

Test material form:

liquid

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

In chemico test system

Details on the study design:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 46.54 mg for both the cysteine and lysine reactivity assay
- Purity: 98%

SOLVENT USED: Acetonitrile (ACN)

POSITIVE CONTROL USED
- Solutions for Cysteine Reactivity Assay: 750 µL Stock solution of 0.667 mM SPCC (Synthetic Peptide Containing Cysteine) + 200 µL ACN + 50 µL Cinnamic aldehyde solution (100 mM in ACN)
- Solutions for Lysine Reactivity Assay: 750 µL Stock solution of 0.667 mM SPCL (Synthetic Peptide Containing Lysine) + 250 µL Cinnamic aldehyde solution (100 mM in ACN)

CO-ELUTION CONTROL (CC):
- Solutions for Cysteine Reactivity Assay: 750 µL Phosphate buffer pH 7.5 + 200 µL ACN + 50 µL test solution (100 mM)
- Solutions for Lysine Reactivity Assay: 750 µL Ammonium acetate buffer pH 10.2 + 250 µL test solution (100 mM)

REFERENCE CONTROL
- Solutions for Cysteine Reactivity Assay: 750 µL of the 0.667 mM SPCC stock solution + 250 µL ACN.
- Solutions for Lysine Reactivity Assay: 750 µL of the 0.667 mM SPCL stock solution + 250 µL ACN.

NUMBER OF REPLICATES
Co-elution control (CC) samples: 1 for both the cysteine and lysine reactivity assay
Positive control samples: 3 each for both the cysteine and lysine reactivity assay
Test item: 3 each for both the cysteine and lysine reactivity assay
Reference Control samples: 3 each for both the cysteine and lysine reactivity assay

SAMPLE INCUBATIONS
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 23 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.

Results and discussion

Positive control results:

Cysteine Reactivity Assay
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.1% ± 0.1%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Lysine Reactivity Assay
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 64.8% ± 1.7%. 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

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC depletion%

Value:

100

Positive controls validity:

valid

Remarks on result:

other: the mean SPCC depletion of the test item

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL depletion%

Value:

93.8

Positive controls validity:

valid

Remarks on result:

other: the mean SPCL depletion of the test item

Other effects / acceptance of results:

- Precipitation: No precipitate or phase separation was observed in any of the samples while after incubation, a phase separation was only observed in the SPCC test item samples.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

Any other information on results incl. tables

In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 93.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 96.9% and as a result the test item was considered to be positive in the DPRA and classified in the “highreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Applicant's summary and conclusion

Interpretation of results:

other: positive

Conclusions:

The test item was considered to be positive in the DPRA and classified in the "high reactivity class" when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this study was to determine the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OCED Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After 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 spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were 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.

 

Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples while after incubation, a phase separation was only observed in the SPCC test item samples.

In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 93.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 96.9%.

 

As a result the test item was considered to be positive in the DPRA and classified in the "high reactivity class" when using the Cysteine 1:10 / Lysine 1:50 prediction model.