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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two mutagenicity tests and one QSAR modelling conducted on the test substance: i) a negative in vitro mammalian cell mutagenicity test (OECD 476, EU B.17, GLP, Klimisch 1), and ii) a weakly positive in vitro Ames test in one out of 5 bacterial strains (OECD 471, EU B.13/14, non-GLP, Klimisch 2). The DEREK QSAR modelling rates the genotoxicity ot the test substance as "plausible" (Klimisch 4).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A GLP study in acordance with OECD Guideline 476 and EU B.17 test method.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The objective of this study was to determine whether bis-(2-ethylhexyl)-citraconate test item induces genotoxicity (point mutations and/or gross chromosomal changes) at the thymidine kinase (tk) locus in L5178Y 3.7.2 C mouse lymphoma cells cultured in vitro in the absence and presence of a rat liver metabolising system. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). The principle of this assay is based on placing cells under selective pressure so that only mutant cells are able to survive. Wells containing viable clones are identified by eye using background illumination and counted.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk+/- (thymidine kinase) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection.

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate S9 fraction

Test concentrations with justification for top dose:

Assays 1a and 2 a: 800, 400, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (3 hours with metabolic activation); Assay 1b: 300, 250, 225, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (3 hours without metabolic activation); Assay 2b: 30; 27.5; 25; 22.5; 20; 17.5; 15; 12.5; 10; 7.5 and 5 μg/mL (24 h without metabolic activation)

Vehicle / solvent:

Acetone (vehicle of the test item and negative control).
Dimethyl sulfoxide (DMSO) was used as the vehicle of positive control chemicals in the main experiments. NEgative (vehicle) control cultures for the positive control substances were treated with DMSO only.

Untreated negative controls:

yes

Remarks:

acetone

Negative solvent / vehicle controls:

yes

Remarks:

untreated control sample

True negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Remarks:

NQD without, CP with metabolic activation in DMSO solvent

Details on test system and experimental conditions:

Test system: Mouse lymphoma cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study).

Growth media: Three types of RPMI 1640 medium were prepared.

The post-mitochondrial fraction (S9 fraction) was prepared from rat liver by the Microbiological Laboratory of CiToxLAB Hungary Ltd. The batch of S9 used in this study was found active under the test conditions. For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%. Cultures treated in the absence of S9-mix received 1 mL of 150 mM KCl (except for the 24-hour treatment). Prior to addition to the culture medium, the S9-mix was kept in an ice bath.


A preliminary toxicity test (a 3 hour treatment with/without S-9 mix and a 24-h treatment without the mix) was performed to select dose levels for the main assays.

Main mutation assays:
In Assay 1, cells were treated for 3-hours in the presence and absence of S9 mix. In Assay 2, cells were treated for 3-hours in the presence of S9 mix and for 24-hours in the absence of S9 mix.
The concentrations used in the main tests were as follows: Assays 1a and 2 a: 800, 400, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (3 hours with metabolic activation); Assay 1b: 300, 250, 225, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (3 hours without metabolic activation); Assay 2b: 30; 27.5; 25; 22.5; 20; 17.5; 15; 12.5; 10; 7.5 and 5 μg/mL (24 h without metabolic activation).

A suitable volume* of RPMI-5 medium, vehicle (solvent), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, at least 107 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, at least 4x106 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) (RPMI-5).
*Note: Treatment volume was 0.2 mL for positive control solutions and their solvent controls; and treatment volume was 0.1 mL for test item formulations and its solvent control or RPMI-5 medium.

Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37 °C ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the 3-hour treatments. After the treatment period, cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Measurement of pH and osmolality was also performed after the treatment period.

Where sufficient cells survived, cell density was adjusted to a concentration of 2x105 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 25 mL of suspension) or diluted to be plated for survival.


Plating for survival: Cultures of cell density 2x105 cells/mL, were further diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for about two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Expression period: To allow expression of TK- mutations, cultures were maintained in flasks for approximately 3 days. During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2x105 cells/mL and transferred to flasks for further growth. On completion of the expression period, at least seven concentrations, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

Plating for viability: At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1x104 cells/mL with RPMI-20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for -trifluorothymidine (TFT) resistance: At the end of the expression period, the cell concentration was adjusted to 1x104 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2x103 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the mechanism of action of the test substance.

Evaluation criteria:

Assay acceptance criteria:
The assay was considered valid if all of the following criteria were met (based on the relevant guidelines and M. Moore 2006):
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (50-170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative (vehicle) controls is within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations are present, where the highest concentration produces approximately 80-90% toxicity (measured by %RS or RTG), results in precipitation, or it is 5 mg/mL, 5 μL/mL or 0.01 M (whichever is the lowest), or it is the highest practical concentration.

Evaluation criteria: The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:

Please see the following field.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Additional information in attachment 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Validity of the mutation assays:

The plating efficiencies for the negative (vehicle) control of the test item at the end of the expression period (PEviability) were within the acceptable range (65-120 %) in all assays.

The number of test concentrations evaluated for each treatment was eight in each case, which met the acceptance criteria.

The tested concentration range in the study was considered to be adequate as in those cases where cytotoxicity occurred, the highest examined concentration produced approximately 80-90% toxicity (i.e. approximately 10-20% relative survival or relative total growth) in each case*. In those cases where no cytotoxicity occurred, samples up to the solubility limit were examined. Lower test concentrations were evenly spaced by a factor of no more than two.
*Note: In Assay 1, in case of the experiment without metabolic activation, the relative survival value of the highest surviving concentration of 250 μg/mL was 16%, while the relative total growth value was 1%. As both data indicated cytotoxicity, this concentration was selected as highest evaluated concentration, although the relative total growth in this case showed stronger cytotoxicity. This fact was considered not to adversely affect the results of the study; furthermore, there was a good correlation between the relative survival and relative total growth value of the highest evaluated concentration of 25 μg/mL (8 and 12%, respectively) in the experiment without metabolic activation of Assay 2.

The overall study was considered to be valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

PRELIMINARY EXPERIMENT: Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment was 4000 μg/mL (approximately 0.01M concentration). Tabulated results of the preliminary experiment are given in Attachment 1 (Appendix 15).

Insolubility and cytotoxicity were observed in the preliminary experiment. Concentrations for the main experiments were selected to cover the range from cytotoxicity to little or no cytotoxicity, or up to the solubility limit according to the instructions of the relevant OECD guideline. Lower test concentrations were separated by factor of two, but more closely spaced concentration were examined in the expected cytotoxic range. At least eleven concentrations were selected for the main experiments.

MUTATION ASSAYS In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel sub-cultured without test item for approximately 3 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 800, 400, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with metabolic activation); and 300, 250, 225, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment without metabolic activation).

Data are presented for survival (Appendices 2 and 5), viability (Appendices 3 and 7) and mutagenicity (Appendices 4, 9 and 11). Results of the visual examination after treatment, as well as pH and osmolality data are presented in Appendix 13.

In Assay 1, insolubility was detected in the final treatment medium at the end of the treatment in the 800-150 μg/mL concentration range with metabolic activation and in the 300-150 μg/mL concentration range without metabolic activation. There were no large changes in pH or osmolality after treatment. In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed in the tested concentration range (results are shown in Table 1 of Appendix 2). Therefore, an evaluation was made using data of the eight highest examined concentrations (800-75 μg/mL). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis (for more details see Table 9 of Appendix 4).

In the absence of S9-mix (3-hour treatment), excessive cytotoxicity of the test item was observed at 300 μg/mL concentration, only a few cells of this sample survived the expression period (as detailed in Table 2 of Appendix 2). An evaluation was made using data of the next concentration of 250 μg/mL (relative survival value of 16%) and seven lower concentrations (a total of eight concentrations in the 250-12.5 μg/mL concentration range). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis (for details see Table 10 of Appendix 4).

In Assay 2, metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 800, 400, 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with metabolic activation); and 30; 27.5; 25; 22.5; 20; 17.5; 15; 12.5; 10; 7.5 and 5 μg/mL (experiment without metabolic activation).

Data of Assay 2 are presented for survival (Appendices 2 and 6), viability (Appendices 3 and 8) and mutagenicity (Appendices 4, 10 and 12). Results of the visual examination after treatment, as well as pH and osmolality data are presented in Appendix 13. In Assay 2, similarly to the first experiment, insolubility was detected in the final treatment medium at the end of the treatment in the 800-150 μg/mL concentration range with metabolic activation. No insolubility was detected in the experiment without metabolic activation. There were no large changes in pH or osmolality after treatment. In the presence of S9-mix (3-hour treatment), similarly to the first test, no cytotoxicity was observed (detailed results are shown in Table 3 of Appendix 2). Therefore, an evaluation was made using data of the eight highest examined concentrations (800-75 μg/mL). No biologically relevant or statistically significant increase in the mutation frequency was observed. No dose response to the treatment was indicated by the linear trend analysis (for more details see Table 11 of Appendix 4). In the absence of S9-mix (24-hour treatment), excessive cytotoxicity was observed at 30 and 27.5 μg/mL concentrations, cells of these samples died during the expression period (results are shown in Table 4 of Appendix 2). An evaluation was made using data of the next concentration of 25 μg/mL (relative total growth value of 12%) and the following seven lower concentrations (a total of eight concentrations in the 25-7.5 μg/mL concentration range). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis (details are shown in Table 12 of Appendix 4). Some minor increases in the mutation frequency were observed sporadically in Assays 1 and 2; however, they were without any statistical significance and the difference between the observed values and the relevant solvent control value did not exceed the global evaluation factor, so they was considered as biologically not relevant increases, just showing the biological variability of the test system.

Conclusions:

Interpretation of results (migrated information):
negative with or without metabolic activation

Treatment with the test item did not result in a statistically significant or biologically relevant increase in mutation frequency in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. Therefore, no mutagenic activity of the test item was observed in the performed experiments.

Executive summary:

The Mouse Lymphoma Assay with the test material on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study. It was conducted according to OECD 476 and EU B.17 under GLP and rated as Klimisch 1. Treatment with the test item did not result in a statistically significant or biologically relevant increase in mutation frequency in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. Therefore, no mutagenic activity of the test item was observed in the performed experiments and the result is assessed as negative evidence in classification and labelling and PBT assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The Mouse Lymphoma Assay with the test material on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study. It was conducted according to OECD 476 and EU B.17 under GLP (Klimisch 1). Treatment with the test item did not result in a statistically significant or biologically relevant increase in mutation frequency in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. Therefore, no mutagenic activity of the test item was observed in the performed experiments and the result is assessed as negative. The study was selected as the key for classification and labelling and PBT assessment.

The test item had weak mutagenic activity in one Salmonella typhimurium strain out of 4 tested strains (TA98, TA100, TA1535 and TA1537 and one Escherichia coli strain WP2 uvrA) under the test conditions used in this study (OECD 471, EU B.13/14 and EPA OPPTS 870.5100 screening test, non-GLP, Klimisch 2). The observed mutation factor 2.19 was slightly over the biologically relevant threshold limit 2 in the strain TA98, and the result is therefore assessed as weakly positive and used as supporting for classification and labelling and PBT assessment.

The potential of bis-(2-ethylhexyl)-citraconate to cause chromosome damage in vitro in mammalian cells was rated as "plausible" in DEREK modelling (Klimisch 4). The expert assessment concludes that the alert for genotoxicity is generally restricted to in vitro mammalian cell assays, and is not normally applicable to in vivo situations. This alert is considered to be unlikely to be of concern for in vivo studies or human health.

Conclusion: based on the negative in vitro mouse lymphoma assay (OECD 476, EU B.17 under GLP, Klimisch 1) the test substance is assessd as non-genotoxic, even if there is weak evidence of genotoxicity in the supporting bacterial mutation test (non-GLP OECD 471, Klimisch 2) and DEREK modelling (Klimisch 4, weight of evidence).

Justification for selection of genetic toxicity endpoint
One negative Klimisch 1 key study in vitro, a weakly positive bacterial mutation test (in vitro, Klimisch 2) used as WoE and a "plausible" DEREK modelling result (Klimisch 4).

Justification for classification or non-classification

The test substance is assessed as non-genotoxic based on a negative in vitro mouse lymphoma assay (OECD 476, EU B.17 under GLP, Klimisch 1). There is weak positive evidence in bacterial mutation test (OECD 471, non-GLP) and DEREK modelling (both Klimisch 2).