2-Butenedioic acid, 2-methyl-, 1,4-bis(2-ethylhexyl) ester, (2Z)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP in chemico study, ECVAM recommends use as weight of evidence only.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Test method evaluated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM, November 2013).

Principles of method if other than guideline:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides in the DPRA Assay. The result was applied for skin sensitization assessment.

GLP compliance:

no

Remarks:

The laboratory had CLP certificate. SOPs of the laboratory were followed.

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

EC Number:

807-715-4

Cas Number:

1354569-12-2

Molecular formula:

C21 H38 O4

IUPAC Name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

Test material form:

other: liquid

In vivo test system

Test animals

Species:

other: none

Strain:

other: none

Details on test animals and environmental conditions:

A diluted solution of cysteine or lysine was incubated with the test item at the ratios 1:10 cysteine: test item and 1:50 lysine: test item for 24 hours.
Each chemical (test item or positive control) was pre-weighed and stored under appropriate conditions until ready to perform testing.
The positive control was dissolved in acetonitrile at 100 mM. The test item was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation was a colorless liquid. Each formulation was prepared within 1 hour before use.

The synthetic cysteine peptide solution was prepared in an aqueous phosphate buffer (pH 7.5) solution. The detailed preparation method is described in a CiToxLAB France analytical method, specific to the DPRA test.
The lysine peptide solution was prepared in an aqueous ammonium acetate buffer (pH 10.2) solution. Thepreparation method is described in a CiToxLAB France analytical method, specific to the DPRA test.

Study design: in vivo (non-LLNA)

Details on study design:

All samples (co-elution control, QC, test item and positive control samples) were incubated during 24 (± 2) hours at room temperature and protected from light before injection onto the HPLC/UV system.

At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis. No precipitate was observed in the vials incubated with the cysteine peptide, as a result, these vials were directly transferred onto the HPLC/UV system. However, some "micelles" were observed in the vials incubated with the lysine peptide. Therefore these vials and QC sample vials as well, were centrifuged at 400 g for 5 minutes at room temperature. Supernatants were then collected and were transferred onto the HPLC/UV system.

Positive control substance(s):

yes

Remarks:

cinnamaldehyde, CAS 14731-10-9

Results and discussion

Positive control results:

After the analysis of the co-elution sample chromatograms, the test item was found to not co-elute either with the lysine or the cysteine peptides.
Mean percentage depletion values were calculated for each peptide using the formula described above.

In vitro / in chemico

Results

Results 1

Parameter:

other: Migrated information from in vitro study

Remarks on result:

other: Reading: other: Mean depletion value citraconate 0.24 %. . Hours after challenge: 24.0. Group: other: lysine peptide. Dose level: 1:50 lysine:test item in acetonitrile. Total no. in groups: 3.0. Clinical observations: SD 0.2, % CV -70.6.

Results 2

Parameter:

other: Migrated information from in vitro study

Remarks on result:

other: Reading: other: Mean depletion value citraconate 4.22 %. . Hours after challenge: 24.0. Group: other: cysteine peptide. Dose level: 1:10 cysteine:test item in acetonitrile. Total no. in groups: 3.0. Clinical observations: SD 0.58, % CV 13.28.

Results 3

Parameter:

other: Migrated information from in vitro study

Remarks on result:

other: Reading: other: Mean depletion rate citraconate 1,99 %. . Hours after challenge: 24.0. Group: other: Mean of lysine and cysteine scores.

Results 4

Parameter:

other: Migrated information from in vitro study

Remarks on result:

other: Reading: other: Mean depletion value control (cinnamanaldehyde) 65.54 %. . Hours after challenge: 24.0. Group: other: lysine peptide. Dose level: 100 mM in acetonitrile. Total no. in groups: 3.0. Clinical observations: SD 0.8, % CV 1.3.

Results 5

Parameter:

other: Migrated information from in vitro study

Remarks on result:

other: Reading: other: Mean depletion value control (cinnamanaldehyde) 100 %. . Hours after challenge: 24.0. Group: other: cysteine. Dose level: 100 mM in acetonitrile. Total no. in groups: 3.0. Clinical observations: SD 0, % CV 0.

Results 6

Parameter:

other: Migrated information from in vitro study

Remarks on result:

other: Reading: other: Mean depletion rate control (cinnamanaldehyde) 81.27 %. . Hours after challenge: 24.0. Group: other: Mean. Dose level: 100 mM in acetonitrile.

Any other information on results incl. tables

Original result tables for the test item and for the positive control (including the calibration data and quality controls) are presented in Attachment 2.

The mean depletion values were: -0.24% for the lysine peptide, 4.22% for the cysteine peptide.

The mean of the percentage cysteine and percentage lysine depletions was calculated to be 1.99%.

Accordingly, the test item was considered to have no/minimal peptide reactivity (< 6.38 % criterion).

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: ECVAM recommendation 2013

Conclusions:

The mean depletion values were: -0.24% for the lysine peptide, 4.22% for the cysteine peptide. The mean of the percentage cysteine and lysine depletions was calculated to be 1.99%. Compared to the method criterion 6.38 %, the test item was assessed to have no/minimal peptide reactivity.

Executive summary:

The reactivity of bis (2 -ethylhexyl)citraconate was evaluated in chemico in a DPRA Assay (EURL ECVAM 2013) by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The result was applied for skin sensitization assessment. The mean depletion values were -0.24% for the lysine peptide and 4.22% for the cysteine peptide. The mean of the percentage cysteine and lysine depletions was calculated to be 1.99%. Compared to the method criterion 6.38 %, the test item was assessed to have no/minimal peptide reactivity, suggesting no/minimal potential to cause skin sensitization.

The result is used as weight-of-evidence for classification and labelling of the substance (Klimisch 2), in line with the ECVAM recommendation.