Tributyl benzene-1,2,4-tricarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From October 16,2007 to February 15,2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant study performed in accordance with internationally recognised test methods. For read across justification see Section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

EC Number:

222-020-0

EC Name:

Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Cas Number:

3319-31-1

IUPAC Name:

tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Details on test material:

- Name of test material (as cited in study report):DIPLAST TM/MG
- Molecular formula (if other than submission substance): C33 H54 O6
- Molecular weight (if other than submission substance): 546.79
- Smiles notation (if other than submission substance): c1(c(ccc(c1)C(OC[C@@H](CCCC)CC)=O)C(OC[C@@H](CCCC)CC)=O)C(OC [C@@H](CCCC)CC)=O
- InChl (if other than submission substance): 1S/C33H54O6/c1-7-13-16-25(10-4)22-37-31(34)28-19-20-29(32(35)38-23-26 (11-5)17-14-8-2)30(21-28)33(36)39-24-27(12-6)18-15-9-3/h19-21,25-27H,7-18,22- 24H2,1-6H3
- Structural formula attached as image file (if other than submission substance): see 3319-31-1 structure.png

- Physical state:colourless liquid
- Lot/batch No.:2117207170
- Analytical purity:97.37 %
- Storage condition of test material:room temperature:

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518).

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

78.1, 156, 313, 625, 1250 and 2500 µg/mL used in the cytogenetic assay.

Vehicle / solvent:

- Solvent used: Ethanol

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9

Details on test system and experimental conditions:

S9 metabolising system
S9 tissue homogenate was prepared from the livers of five young male Sprague-Dawley rats which had received treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic metabolising enzymes.

Preparation of test cell cultures
A cell suspension (1E6 cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures. The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and in the presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in PBS, cells were resuspended in 20 ml RPMI minimal medium. Cell concentrations were adjusted to 8 cells/ml using complete medium (20%) and, for each dose level, 0.2 ml was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
Treatment of cell cultures
Experiments were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in this experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a long treatment time (24 hours).
After washing in PBS, cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2E5 cells/ml. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Determination of survival
Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/ml using complete medium (20%). A 0.2 ml aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. After incubation, wells containing viable clones were identified by eye using background illumination and then counted.

Expression period
During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2E5 cells/ml.

Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/ml) and an estimated 2E3 cells were plated in each well of four 96-well plates.
Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified . In addition, the number of wells containing large colonies and the number containing small colonies were scored.

Plating for viability
After dilution, in complete medium (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Evaluation criteria:

The assay was considered valid if the following criteria were met:

(i) The cloning efficiencies at Day 2 in the untreated control cultures in the absence of S9 metabolic activation fell within the range of 65-120%.

(ii) The untreated control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8 – 32.

(iii) The mutant frequencies in the untreated control cultures fell within the range of 50 200 x 106 viable cells.

(iv) The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

See "any other information" for details

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Cytotoxicity test
Both in the absence and presence of S9 metabolic activation, the test substance was assayed at a maximum dose level of 5000 µg/ml and at lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml.
Slight decreases in relative survival were observed at intermediate dose levels, both in the absence and presence of S9 metabolic activation, using a 3 hour treatment time. No relevant toxicity was observed using a long treatment time in the absence of S9 metabolic activation.

Mutation assays
Two independent assays for mutation to trifluorothymidine resistance were performed.
The mutant frequencies in the solvent control cultures fell within the normal range (5E7 2E8 viable cells). The positive control chemicals induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65-120% and the control growth factor over 2 days fell within the range of 8 – 32 in both experiments.
In the absence of S9 metabolic activation, using the 3-hour treatment time, slight toxicity was observed at the highest dose level (2500 µg/ml) reducing survival to 72% of the concurrent negative control value. The relative total growth was reduced to 78% at the same concentration. Using a long treatment time, no relevant toxicity was observed at any dose level.
In the presence of S9 metabolic activation, no relevant toxicity was observed in the first experiment, while in the second experiment, a slight decrease in relative total growth (60%) was observed only at the intermediate dose level of 313mg/ml.

In Experiment 1, in the presence of S9 metabolic activation, the heterogeneity between replicate cultures for survival at the end of treatment, at the concentration of 625 µg/ml, was higher than usual and thus excluded from the determination of overall heterogeneity. In addition, in the second experiment, in the absence of S9, a slight but statistically significant difference between replicate plates was observed for culture A treated at 625 µg/ml.These results have not affected the validity of the study.

No statistically significant increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test substance at any concentration level.

Osmolality and pH
The test substance did not have any obvious effect on the osmolality or pH of the treatment medium.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that DIPLAST TM/MG does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The substance has been examined for mutagenic activity by assaying for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 

The substance does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions