Ester reaction products of 1,4-Benzenedicarboxylic acid with C11-14 iso-alcohols, C13-rich

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:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented study report which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

Genetic Toxicology Testing of Di(2-ethylhexyl) Terephthalate

Author:

Barber, ED

Year:

1994

Bibliographic source:

Environmental and Molecular Mutagenesis 23:228-233

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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:

di-2-ethylhexyl terephthalate (DEHT)

IUPAC Name:

di-2-ethylhexyl terephthalate (DEHT)

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) terephthalate

EC Number:

229-176-9

EC Name:

Bis(2-ethylhexyl) terephthalate

Cas Number:

6422-86-2

Molecular formula:

C24H38O4

IUPAC Name:

bis(2-ethylhexyl) terephthalate

Details on test material:

Di-2-ethylhexyl terephthalate (DEHT) (Eastman 168 plasticizer; CAS 6422-86-2) is a clear, viscous liquid used as a general purpose plasticizer for flexible polyvinyl chloride (PVC) and polyvinyl chloride/vinyl acetate (PVC/VA) copolymers.

Method

Target gene:

HGPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

A culture of CHO-K1 , subclone BH4, was obtained from the laboratory of Dr. A.W. Hise (Oak Ridge National Labortory, Oak Ridge, TN). Stock cultures were stored in liquid nitrogen and expanded periodically in HAT medium to select for the HGPRT+ phenotype.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Dose levels of 1.25, 2.5, 5, 10, 20 nl/ml were chosen based on cytotoxicity assays. The highest dose level, 20nl/ml, is approximately two times higher than the solubility limit of DEHT in Ham's F12 culture medium containing 1% DMSO.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

medium only

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

0.25mg/ml dimethylnitrosamine

Positive control substance:

N-dimethylnitrosamine

Details on test system and experimental conditions:

Doses were chosen following a cytotoxicty in Ham’s F12 culture medium. Using toxicity relationship as a guide, five concentrations were selected covering the range from 0 to 90% reduction of colony forming ability.

CHO-K1-BH1 cells were seeded into flasks. After 24h incubation, media containing the appropriate concentrations of the test article and the positive and negative (solvent) controls were applied to the flasks and incubated for 4 hours. Following overnight incubation in F12 culture medium, the monolayers were trypsinized, and the cells were reseeded at densities of ~100 cells per flask. These flasks were incubated for 10 days to determine cell survival from the treatments. Cells for mutant scoring were seeded in flasks and subcultured for a period of 9 days to allow for expression of induced mutations. At the end of the expression period, cultures were reseeded in F12 medium containing 6-thioguanine as the selective agent. After 7 days incubation, colonies were stained and counted. The mutant frequency at each treatment condition was calculated by dividing the total number of mutant colonies by the product.

During the assay samples of Hams F12 growth medium containing DEHT were submitted for analysis to confirm dose levels. Analysis of these samples showed good agreement with expected values for the nonactivation portion of the study by values much less than expected for the S-9 containing samples. A stability study showed that 95% of the DEHT could be recovered by extraction 0.5hr after sample preparation but that only 47% could be recovered at 5.0 hr when S-9 was present. Such decomposition of DEHT was not noted when the S-9 activation fraction was omitted. It seems probably that the esterases found in the rat liver S-9 were hydrolyzing the DEHT.

Evaluation criteria:

At the end of the expression period, cultures were reseeded in F12 medium containing 6-thioguanine as the selective agent. After 7 days incubation, colonies were stained and counted. The mutant frequency at each treatment condition was calculated by dividing the total number of mutant colonies by the product.

Statistics:

Mutant frequency data were evaluated using the statistical tables provided by Kastenbaum and Bowman.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high doses during hte treatment phase of the assay

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: 15 doses of DEHT from 0.001 to 20.0 nl/ml were tested in the clonal cytotoxicity assay. Cell survivals from 88.6%-106% were observed at these dose levels, indicating that there was little or no reduction in cloning efficiency. The highest dose level, 20nl/ml is approximately two times higher than the solubility limit of DEHT in Ham’s F12 culture medium containing 1% DMSO (solvent for DEHT). On the basis of these clonal cytotoxicity data, dose levels were chosen.

COMPARISON WITH HISTORICAL CONTROL DATA:
ADDITIONAL INFORMATION ON CYTOTOXICITY: During the treatment phase of the mutagenicity assay, survival-to-treatment data were obtained at the dose levels of DEHT chosen for the assay. At dose levels of 20 and 10nl/ml DEHT in the non-activation assay, the relative cell survival values were reduced to 69.2% and 72.9% respectively, indicating a weak toxic response had occurred. The positive control, ethyl methanesulfonate at 0.25mg/ml, reduced the relative cell survival to 40.4% in this portion of the assay. The activation portion of the assay, the positive control chemical, dimethylnitrosamine at 0.25mg/ml, reduced the relative cell survival to 34.8%. All other treatments in both the non-activation and the activation portions of the assay resulted in relative survivals not different from the untreated controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The relative cloning efficiencies for all the treatments were near 100%, indicating good cell survival in the assay. The mutant frequency data for the DEHT-treated cultures and for the negative and solvent control cultures for the nonactivation assay lie between 0.5 and 2.9 mutants per million surviving cells. The mutant frequency for the ethyl methanesulfonate treated cells is 298.8 per million surviving cells, a very large and significant increase above the negative controls. The data form the DEHT-treated cultures do not show any statistically significant increases when compared with the negative controls. The mutant frequency values for the S-9 activation portion of the study are somewhat larger than those in the table; however the negative control values are within the limits described for this assay and are consistent with the historical values for this laboratory. There are no statistically significant increases among the DEHT-treated cell populations. The positive control data show a significant increase in mutant frequency when compared to the medium or solvent controls. 

Applicant's summary and conclusion

Conclusions:

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

The test material, does not produce signification increased in mutant frequency in the CHO cells and is therefore negative in this mammalian cell mutagenicity test. This finding does not warrant the classification of the submission substance as a germ cell mutagen under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Executive summary:

DEHT was tested in the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) in vitro mammalian cell mutagenicity assay. This assay system detects forward mutations f the X-linked HGPRT locus using the toxic purine analog 6-thioguanine as the selective agent. The lack of a positive response in the cultures treated with DEHT at dose levels up to and slightly higher than the solubility limit of DEHT in the assay medium demonstrates that DEHT was not mutagenic in this test system.