Diethyl phthalate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 19,2010 to July 06,2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well described GLP compliant study conducted to recognized international test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl phthalate

EC Number:

201-550-6

EC Name:

Diethyl phthalate

Cas Number:

84-66-2

Molecular formula:

C12H14O4

IUPAC Name:

.

Details on test material:

- Name of test material: Diethyl Phthalate (DEP)
- IUPAC name: 1,2-Benze​nedicarbo​xylic aci​d diethyl​ ester
- Molecular formula : C12H14O4
- Molecular weight : 222.2372 g/mol
- Smiles notation : c1(c(C(OCC)=O)cccc1)C(OCC)=O
- InChl : 1/C12H14O4/c1-3-15-11(13)9-7-5-6-8-10(9)12(14)16-4-2/h5-8H,3-4H2,1-2H3
- Physical state: Clear colourless liquid
- Analytical purity: 99.96 % (as ester content)
- Lot/batch No.: H126210019
- Expiration date of the lot/batch: 15 February 2013
- Storage condition of test material: Ambient temperature, protected from humidity

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 were obtained from American Type Culture Collection

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Assay N° S9 Treatment Dose level (µg/ml)Treatment
Time (hours)
1 - 3 578, 525, 477, 434, 395 and 359
1 + 3 925, 771, 578, 289, 145 and 72.3
2 - 24 444, 389, 333, 278, 222 and 111
2 + 3 925, 881, 839, 799, 761 and 725

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

absence of S9 metabolic activation system

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

in presence of S9 metabolic activation system

Details on test system and experimental conditions:

Preparation of test cell cultures:

A cell suspension (1 x 10^6 cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.
The treatment media were prepared as follows:

Without S9 metabolism – 3-hour treatment time

Cell suspension (1 x 10^6 cells/ml in complete medium 5%) 10.0 ml
Complete medium (5%) 9.8 ml
Control or Test item solution 0.2 ml
20.0 ml
Without S9 metabolism – 24-hour treatment time

Cell suspension (1 x 106 cells/ml in complete medium 10%) 3.0 ml
Complete medium (10%) 16.8 ml
Control or Test item solution 0.2 ml
20.0 ml
With S9 metabolism – 3-hour treatment time

Cell suspension (1 x 106 cells/ml in complete medium 5%) 10.0 ml
S9 mix 9.8 ml
Control or Test item solution 0.2 ml
20.0 ml

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 were used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and 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 Phosphate Buffered Saline (PBS), cells were resuspended in 20 ml of complete medium (10%). 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 7 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 solvent and positive controls, in the absence and presence of S9 metabolising system. The results presented for the first experiment both in the absence and presence of S9 metabolism, were obtained in a repeated experiment. In the original experiment the mutation frequency and the cloning efficiency of the negative control cultures fell outside the range of acceptability. Data are not presented in this report but are retained in the Study File and archived as described in the Study Protocol.
Preparation of test cell cultures was performed as described . Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
The cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 105 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 cells/ml using complete medium A (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 7-8 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 2 x 105 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 2 x 103 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 12-13 days and wells containing clones were identified as described in section 4.3.2 and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability

After dilution in complete medium A (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 12-13 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).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH:The addition of the test item solution did not have any obvious effect on the pH of the treatment medium in the absence of S9 metabol ic activation, while it generated a slight dose dependent decrease over the concurrent negative control in the presence of S9 metabolism. The obse rved values however were within an acceptable range.
- Effects of osmolarity:. The addition of the test item solution did not have any obvious effect on the osmolality of the treatment medium.
- Solubility:The test item was found to be soluble in DMSO and ethanol at a dose level of 222 mg/ml. This level permitted a final concentration in the t reatment medium of 2220 µg/ml corresponding to 0.01 M
- Precipitation:no precipitation of the test item was noted at the beginning or at the end of the treatment incubation period at any concentration test ed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the absence of S9 metabolic activation, using a short and long treatment time, no statistically significant increases in mutant frequency were observed.

 

In the presence of S9 metabolic activation, in the first experiment, a statistically significant increase in mutation frequency was observed at the highest analysable concentration of 771 μg/ml (p<1%) and a significant dose‑relationship was indicated by the linear trend analysis (p<0.1%). In the second experiment, using a modified dose-range, a statistically significant increase in mutant frequency was observed only at an intermediate concentration (799 μg/ml; p<5%). A significant dose-relationship was indicated by the linear trend analysis (p<5%). However, the mutant frequencies observed at these dose levels were within the historical control range observed at the testing facility and the induced mutant frequencies (IMF) were lower than the global evaluation factor (GEF) suggested for the microwell method (126 x 10^‑6). Hence, the linear trend and the observed increases were considered to be of no biological significance.

 

For the negative and positive controls, the number of wells containing small colonies and those containing large colonies were scored.

The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. Recovery of small colony mutants was observed following treatment with the positive controls.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Diethyl Phthalate (DEP) 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:

Mutagenic activity has been investigated 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. Diethyl phthalate does not induce mutation at the TK locus of L5178Y mouse lymphoma cellsin vitroin the absence or presence of S9 metabolic activation.