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

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

2 (reliable with restrictions)

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: Stemina SOPs

Principles of method if other than guideline:

Metabolomics study: The teratogenicity potential ot the test material was assessed based on the metabolic perturbation observed following non-targeted metabolomic analysis of spent cell culture media from human embryonic stem (hES) cells exposed to the compound. Applicable SOP’s within Stemina’s Quality Management Plan were followed.

GLP compliance:

no

Type of method:

in vitro

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

Test animals

Species:

other: human

Strain:

other: embryonic stem (hES) cells

Details on test animals or test system and environmental conditions:

WA09 hES cells

Administration / exposure

Route of administration:

other: in vitro plates

Vehicle:

DMSO

Remarks:

0.1 %

Details on exposure:

Cell viability and dose selection:
Cell viability assays were performed to establish three concentrations for hES cell dosing for metabolomics experiments. First, WA09 hES cells were plated in 96-well format at 100,000 cells per well and treated with nine concentrations of the compound ranging from 0.04μM - 300μM. The compound was initially dissolved in DMSO to create stock solutions and then serially diluted in mTeSR1 media for treatment. The final concentration of DMSO was 0.1% for all treatments. Compound exposure began approximately 24 hours after plating. hES cells were exposed to the test compound for 72 hours, with media and test compound replacement every 24 hours. At the end of the treatment period, cell viability was assessed using the CellTiter-Fluor cell viability assay (Promega, Fitchburg, WI, USA). The relative fluorescence units (RFU) for viability (live cell protease) were normalized to the 0.1% DMSO control readout by dividing the viability RFU of each well by the average 0.1% DMSO viability RFU.

Dose-response curves were constructed to determine three concentrations for metabolomics analysis. In keeping with the general aim of this study, doses were selected which were not likely to reflect major metabolic or biochemical injury as a result of cytotoxicity or cell death. Since no change in cell viability was observed, the three concentrations chosen were 10 fold serial dilutions of the highest exposure level from the dose response assay (3, 30, and 300 μM).

Cell Culture for Metabolomic Analysis:
For metabolomic analysis, hES cells were dosed at three selected concentrations (3, 30, and 300μM). Each 96-well plate included media controls with and without test compound, 0.1% DMSO solvent control cells and cells exposed to the three levels of the test compound. (Additionally, to confirm accuracy of predictions), a positive and negative control was included on the plate. Compounds used as positive and negative controls were busulfan (5.3 μM) and doxylamine (0.38 μM), respectively.

For metabolomics treatment, WA09 hES cells were plated in 96-well plates at 100,000 cells per well. Each treatment was performed with 6 replicates. Each compound stock solution was made in DMSO and each final solution used to dose hES cells contained 0.1% DMSO. The spent media from the final 24-hours of treatment were collected (150μL) and added to acetonitrile containing 13C6 labeled arginine and hippuric acid as internal standards (final acetonitrile concentration 40%). Quenched samples were stored at -80°C until they were prepared for metabolomic analysis. Cell viability was measured after sample collection using the CellTiter-Fluor viability assay and used as a quality control check. Quality criteria for the assay require that if values within a treatment show a coefficient of variation greater than 10%, outlier tests were performed and the data removed. If more than one data point was outside this quality criterion, the experiment was repeated.

Sample Preparation:
Millipore Multiscreen Ultracel-10 molecular weight cut-off plates were used for isolation of small molecular weight compounds (<10 KDa) carried forward into metabolomics analysis. Prior to use the plates were first washed once with a 0.1% sodium hydroxide solution and then twice with LC-MS grade water to remove a known residual contaminant polymer. The acetonitrile-quenched samples were added to the washed filter plates then centrifuged at 2000xg for approximately 200 minutes at 4 oC. The filtrate was then collected and dried completely using a SpeedVac concentrator system. Lastly, the dried samples were reconstituted in 52.5 μL of a 1:1 solution of 0.1% formic acid in water: 0.1% formic acid in acetonitrile, which contained two additional internal standards: 13C-ornithine and 13C-cystine.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

LC-MS Experimental:
Samples were analyzed using two Agilent Q-TOF 6500 series mass spectrometers each coupled with an Agilent 1290 HPLC systems via an electrospray (ESI) interface. Data acquisition was performed with MassHunter Acquisition software using high-resolution exact mass conditions. The mass resolution of the instrument (m/z dependent on TOF systems) was approximately 3,000–12,000. Gradient time 0 - 23 hours, flow rate 0.5 - 1.0 mL/min.

As part of the standard quality control program an equilibration injection was made at the beginning of each 96 well plate run and all samples were randomized with extraction blanks interspersed.

Duration of treatment / exposure:

Following compound exposure for 72 hours, viability was ascertained via the CellTiter-Fluor assay to determine cytotoxicity (Figure 3).
The data from this experiment was used to determine appropriate concentrations for metabolomics treatments.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
3
Basis:
other: µM

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30
Basis:
other: µM

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300
Basis:
other: µM

No. of animals per sex per dose:

6 replicates were used for metabolomics treatment.

Details on study design:

Metabolomics: Each 96-well plate included media controls with and without test compound, 0.1% DMSO solvent control cells and cells exposed to the three levels of the test compound. (Additionally, to confirm accuracy of predictions), a positive and negative control was included on the plate. Compounds used as positive and negative controls were busulfan (5.3 μM) and doxylamine (0.38 μM), respectively.

Statistics:

Data Processing: The data processing paradigm is outlined in Figure 1. Data preprocessing was conducted prior to statistical analysis for differential feature assessment. The process began with conversion of Agilent raw data files to mzData open source file format (Agilent Mass Hunter Qual v.B 05.00). The resulting files contain centroid data of deisotoped peaks with an absolute height of greater than 300 counts (above the level of noise).
From these files mass features were created and integrated using the R software package xcms (v. 1.26.1). Mass features were detected using the centWave algorithm and deviations in retention times were corrected using the Obiwarp algorithm. Mass feature intensity is based on Mexican hat
integrated values of extracted ion chromatograms. (Statistical analysis continues below)

Results and discussion

Effect levels

1

Dose descriptor:

other: Teratogenicity prediction

Effect level:

other: NON (0.85) TER (0.15) confidence 0.70

Based on:

other: metabolomics of HES cells

Sex:

male/female

Basis for effect level:

other: Low dose 3 µM (n=6)

2

Dose descriptor:

other: Teratogenicity prediction

Effect level:

other: NON (0.66) TER (0.34) Confidence 0.32

Based on:

other: metabolomics of HES cells

Sex:

male/female

Basis for effect level:

other: Medium dose 30 µM (n=6)

3

Dose descriptor:

other: Teratogenicity prediction

Effect level:

other: NON (0.63) TER (0.37) Confidence 0.26

Based on:

other: metabolomics of HES cells

Sex:

male/female

Basis for effect level:

other: High dose 300 µM (n=6)

4

Dose descriptor:

other: Teratogenicity prediction

Effect level:

other: NON (0.31) TER ((0.69) Conf 0.38

Based on:

other: Metabolomics of HES cells

Sex:

male/female

Basis for effect level:

other: Positive control (n=6)

5

Dose descriptor:

other: Teratogenicity prediction

Effect level:

other: NON (0.68) TER (0.32) Conf. 0.36

Based on:

other: Metabolomics of HES cells

Sex:

male/female

Basis for effect level:

other: Negative control (n=6)

Observed effects

Cytotoxicity with CellTiter-Fluor assay: As shown in Figure 3 the compound showed no dose-response, even at the highest level (300μM).
The data from this experiment was used to determine appropriate concentrations for metabolomics treatments as summarized in Table 2. As the compound showed no cytotoxicity dose-response, the standard screening paradigm for the assay was applied.

Prediction of Teratogenicity: The metabolomics based devTOX Discovery assay indicated that bis(2-ethylhexyl) citraconate showed no signal of teratogenic potential over the exposure range from 3-300 μM, as summarized in Table 3. Predictions are considered conclusive in this assay if the confidence level is 0.1 or greater and all exposure levels tested met this criterion. Evaluation of the control samples suggested the model was
performing within quality specifications in all data sets by correctly predicting the positive and negative controls (busulfan, and doxylamine).

Cytotoxicity: Results of cytotoxicity assays (Figure 4) conducted in support of the metabolomics experiments were consistent with the dose ranging data. No significant cell death was observed in cultures exposed to bis(2-ethylhexyl) citraconate at any of the exposure levels. Positive and negative controls and the DMSO control all responded as expected. All assay results were within quality control specifications (<10% CV).

Any other information on results incl. tables

Significantly Changed Features and Perturbed Pathways: Metabolomics data were analyzed as described in the methods section. A total of two significant changes in cellular metabolism were seen as a results of exposure to bis(2-ethylhexyl) citraconate. Neither mass feature could be assigned putative annotations when queried against the Stemina database, thus they are reported in term of their mass to charge ratios as measured by LC/MS. The low exposure level (3μM) showed one significantly changed mass feature at m/z 158.1531 while the high exposure level (300μM) showed a single significant change at m/z 192.1589. The medium exposure level (30μM) showed significant changes in both mass features. Results for each compound level are summarized in Table 4. This table is intended to be representative rather than exhaustive and raw Excel files can be provided for additional data mining. It should be noted that since the significantly changed features not could be assigned putative annotations, the conduct of pathways analysis was precluded. It should also be noted that these changes were detected at the beginning of the chromatographic run and, as such, may not be reliable indicators of changed metabolites.

Multivariate analysis utilizing supervised methods suggests that there may be some perturbations in metabolism as does EIC based evaluation of predictive model based markers. Unsupervised multivariate analysis does not demonstrate class separation suggesting that a very clear difference in treatment versus DMSO is not present in the data as a whole for each exposure level. That so little metabolic perturbation is seen for the compound, it correlates well with the negative finding for teratogenicity at the exposures examines in this study.

Applicant's summary and conclusion

Conclusions:

The test substance bis(2-ethyllhexyl)citraconate showed no teratogenicity potential at 0.04 - 300 µM concentrations in the CellTiter-Fluor viability assay, or at 3, 30 and 300 µM concentrations in futher devTox Discovery or cytotoxicity assays. Statistical metabolonomics data in Significantly Changed Features and Perturbed Pathways analysis found only two significantly changed mass features, and supports the negative findings.

Executive summary:

Through application of the hES based devTOX Discovery assay and associated statistical models the prediction of the potential for developmental toxicity through exposure to bis(2-ethylhexyl) citraconate was made for three treatment levels (3, 30, and 300 μM). Based on these data, the compound did not show metabolic perturbation indicative of the potential to exhibit teratogenicity over the exposure range studied. In addition, the compound showed no cytotoxcity over the exposure range tested in either the dose ranging or metabolomics portions of the study. Only two significantly changed mass features were detected when treated cells were compared with DMSO controls, further indicating the compound exerts little impact on the growth and metabolism of hES cells.

This in vitro metabolomics study was conducted in accordance with Stemina's Standard Operating Procedures under non-GLP (non applicable OECD or EU test methods were yet available). The results are rated as Klimisch 2, and used as weight of evidence for classification and labelling and PBT assessment.