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

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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.

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)

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.

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

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.

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.

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.

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.

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.

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.

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.

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).

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The reactivity of bis (2 -ethylhexyl)citraconate was evaluated in chemico in a DPRA Assay 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 acceptance criteria of the samples for the calibration curve, quality control and positive control were satisfied. The study was therefore considered to be valid (Klimisch 2), and applied as weight of evidence for classification and labelling. 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 percentage lysine depletions was calculated to be 1.99%. The test item was assessed to have no/minimal peptide reactivity, suggesting no/minimal potential to cause skin sensitization.

The TOPKAT 4.5 QSAR model indicates that bis-(2-ethylhexyl)citraconate is in the category of non-sensitizers. An external expert assessment considered the evidence as conclusive. The statistics in the model meets the 5 OECD principles for QSAR models validation. The four principles set by the REACH Annex XI (1.3) are also met. The results are therefore rated as reliable (Klimisch 2), and applied as weight of evidence for classification and labelling.

The DEREK modelling of bis(2 -ethylhexyl)citraconate for skin irritation was rated as plausible, based on substructural features of the molecule. The expert assessment concludes that the skin sensitisation alert was found in 3 databases with 20 structures, where approximately 50% were sensitisers and 50% non-sensitisers, so the reliability of this alert is not high.The reliability is therefore ranked as Klimisch 4 (not assignable).

Conclusion:

As weight of evidence from DPRA study and TOPKAT assessment there is a low likelihood that bis-(2-ethylhexyl)citraconate may be a skin sensitizer.

Migrated from Short description of key information:
The skin sensitisation was assessed with i) an in vitro DPRA Assay suggesting no/minimal skin sensitisation potential, ii) DEREK QSAR modelling as plausible and iii) TOPKAT QSAR modelling as a non-sensitizer.

Justification for selection of skin sensitisation endpoint:
Weight of evidence: an in vitro DPRA study supported by TOPKAT and DEREK models. Two studies are rated as Klimisch 2, and third as Klimisch 4.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Migrated from Short description of key information:
In accordance with REACH Article 18, testing is not required for this type of submission.

Justification for classification or non-classification

The available data from an in vitro test and a QSAR model do not meet the skin sensitizer classification criteria of the CLP regulation.