Bis(2-ethylhexyl) 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Assays for the induction of gene mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in culture

Author:

Myhr B, Bowers L, Caspary WJ

Year:

1985

Bibliographic source:

Progr. Mutat. Res. 5, 555-568

Reference 2

Reference Type:

publication

Title:

Appendix1: Source purification analytical details 10 test chemicals

Author:

Ashby, J., Paton, D.

Year:

1985

Bibliographic source:

Prog. Mutat. Res. 5, 691-698

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phthalate

EC Number:

204-211-0

EC Name:

Bis(2-ethylhexyl) phthalate

Cas Number:

117-81-7

Molecular formula:

C24H38O4

IUPAC Name:

1,2-bis(2-ethylhexyl) benzene-1,2-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): diethylhexylphthalate DEHP
- Source : Fluka
- Purity : >99%

Method

Target gene:

Thymidine Kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Laboratory cultures were grown as suspension cultures in Fischer's medium at 37 °C and routinely had doubling times near 10h. The medium was supplemented with 2 mM L-glutamine, 110 4g/ml sodium pyruvate, 0.05% pluronic F58, antibiotics, and heat-inactivated horse serum (10% by volume).
- Source: Clone 3.7.2C, were obtained from D. Clive, Burroughs Wellcome Co.,
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

induced and induced Fischer 344 male rats weighing approximately 200 g. The induced rats were injected i.p. with Aroclor 1254 in corn oil (500 mg/kg ) at 5 days prior to kill

Test concentrations with justification for top dose:

125, 250, 500, 750, 1000, 2000, 3000, 5000 nl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol 1%

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: ethyl methane sulfonate 250 nl/ml (EMS) or methyl methane sulfonate 5-10 nl/ml (MMS). With S9: 3-Methylcholanthrene 2.5-3.5 µg/ml (MCA) (induced S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days in presence of Trifluorothymidine

SELECTION AGENT (mutation assays): 3µg/ml TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 3 cultures / dose-level

NUMBER OF CELLS EVALUATED: 600 cells/

DETERMINATION OF CYTOTOXICITY
- Method: The mutant frequency (MF) was calculated by dividing the total number of colonies in the set of 3 mutant selection dishes by the total count in the set of 3 nonselective dishes and multiplying this ratio by 2 X 10e-4. The measurement of the toxicity of each treatment was a parameter called the relative total growth (RTG). The RTG for a treated culture was calculated as a growth ratio for the expression period (the increase in tell number divided by the average increase in tell number for the solvent control cultures) multiplied by a cloning efficiency ratio (cloning efficiency divided by the average cloning efficiency of the solvent controls).

Evaluation criteria:

Solvent Control Cultures
1.The cloning efficiency must be in the 50-115% range.
2.At least two acceptable cultures must be available.
3.The average mutant frequency for ail acceptable cultures must be between 15 x 10e-6 and 110 x 10e-6 for negative evaluations. For clearly mutagenic test chemicals, the range is extended to 10X10e-6 to 150x10e-6.
4.A Chi-square test for consistency among the mutant frequencies of the acceptable cultures must be significant at p < 0.05

Positive Control Cultures
1.The cloning efficiency must be in the 10-115% range.
2.The relative total growth must not be less than 1%.
3.At least one acceptable culture must be available.
4.The average mutant frequency for all acceptable cultures must be within the historical range.

Test Chemical Cultures
1.The cloning efficiency must be in the 10-115% range.
2.The relative total growth must not be less than 1 %.
3.The minimum cell density at the end of the expression period is 3 X 105 tells/ml.
4.The test chemical must remain soluble during treatment.
5.The maximum dose is 5 mg/ml for solids and 5 µl/ml for liquids.
6.Each dose level must have two or more acceptable cultures.
7.A Chi-square test for consistency among the mutant frequencies of the acceptable cultures must be significant at p < 0.05.
8.At least three acceptable dose sets must be available, except when no response is obtained and sets are rejected due to precipitation

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DEHP was evaluated as negative in the mouse lymphoma assay with or without the S9 activation system, although significant increases in mutant frequency appeared to occur. All of the tested doses were insoluble because the dispersion characteristics of DEHP were not realized until after the mutation studies. DEHP initially dispersed in the medium and appeared to dissolve at concentrations less than approximately 2000 nl/ml. However, oil droplets soon formed which were not easily observable in suspension cultures in translucent tubes.
After 4 h incubation at 37°C, a suspension of droplets was clearly observable at 100 nl/ml in the collected medium, and microscopic examinations indicated that the solubility limit was approximately 25 nl/ml. Since the lowest tested dose was 125 nl/ml, it is not surprising that the mutant frequencies appeared to fluctuate randomly as a function of dose.
A 1.9-fold increase in mutant frequency was observed for the 1000 nl/ml treatment without S9 in one trial but not in the other trial or at higher doses. 2-fold increases in mutant frequency occurred for the low dose (125 nl/ml) and high dose (5000 nl/ml) for one activation experiment, but the mutant colony counts were not elevated and the responses were caused primarily by a low cloning efficiency for one member of each triplicate dose set.
Additional studies indicated that DEHP toxicity correlated with the number and size of suspended droplets, which in turn was influenced by the method of dispersion.
The evaluation of DEHP should be based on soluble concentrations less than 25 nl/ml, and it was assumed from the current data that such treatments would be relatively nontoxic and not mutagenic.

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

Under these experimental conditions, DEHP is not considered as mutagenic.

Executive summary:

Diethylhexylphthalate (DEHP) was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the a protocol similar to the OECD n° 476 Guideline.

In a mammalian cell gene mutation assay (TK+/-), mouse lymphoma L5178Y cells cultured in vitro were exposed to DEHP in ethanol for 4 hours at concentrations between 125 and 5000 nl/ml in the presence and absence of metabolic activation. Appropriate positive controls were used and showed a statistical increase in mutant colonies. After a 48h rest period, cells were then incubated for 11-12 days for mutagenicity evaluation with trifluorothymidine 3µg/ml.

The evaluation of DEHP should be based on soluble concentrations less than 25 nl/ml, and it was assumed from the current data that such treatments would be relatively non-toxic and not mutagenic.

Under these experimental conditions, DEHP did not induce any increase in mutant colonies and is not considered as mutagenic.