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EC number: 229-176-9 | CAS number: 6422-86-2
Non Genotoxic based on in vitro studies
In an Ames reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 of S. typhimurium were exposed to di (2-ethylhexyl) terephthalate in ethanol at concentrations of 1.0, 10, 100, 1000, or 10000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method. Under the conditions of the test, di (2-ethylhexyl) terephthalate did not induce gene mutations by base pair changes or frameshifts, while positive control substances induced appropriate responses. Di (2-ethylhexyl) terephthalate was considered to be non-mutagenic in this study.
In a mammalian cell cytogenetics assay, Chinese hamster ovary cell (CHO-WBI) cultures were exposed to di (2-ethylhexyl) terephthalate at concentrations of 700, 800, 900, and 1000 nL/mL with and without metabolic activation. Negative, solvent and positive controls were also prepared and tested. Mitotic index was used to assess cytotoxicity and select concentrations for metaphase analysis. Metaphase cells were examined for chromosomal damage. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background in di (2-ethylhexyl) terephthalate-treated cells. It was concluded that di (2-ethylhexyl) terephthalate showed no evidence of clastogenic activity under conditions of this assay at the concentrations tested.
Analysis of samples containing di (2-ethylhexyl) terephthalate in the cell culture medium showed good agreement with expected values in the absence of metabolic activation, but not when metabolic activation was present. Further analysis revealed that di (2-ethylhexyl) terephthalate levels were reduced significantly within 4.5 hours of treatment initiation. It is hypothesized that the esterases found in the rat liver S-9 were hydrolyzing the added di (2-ethylhexyl) terephthalate.
In a mammalian cell gene mutation assay on the HGPRT locus, CHO cells cultured in vitro were exposed to di (2-ethylhexyl) terephthalate at concentrations of 0, 1.25, 2.5, 5, 10, or 20 nL/mL in the absence or presence of metabolic activation (induced rat liver S-9). In the absence of metabolic activation, no cytotoxicity was observed at concentrations up to 20 nL/mL. In the presence of metabolic activation, di (2-ethylhexyl) terephthalate levels were reduced significantly within 4.5 hours and it is hypothesized that the esterases found in the rat liver S-9 were hydrolyzing the added di (2-ethylhexyl) terephthalate. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background for cultures treated with di (2-ethylhexyl) terephthalate even at cytotoxic dose levels.
The mutagenic/genotoxic potential of di (2-ethylhexyl) terephthalate has been characterized in several well-conducted bacterial and mammalian in vitro mutagenicity assays. In two bacterial reverse mutation assays conducted by a method equivalent or similar to OECD Guideline 471, there was no increase in mutation frequency in any strain of Salmonella typhimurium at concentrations up to10000 μg/plate in the presence or absence of metabolic activation and there was no evidence of cytotoxicity at the highest concentration tested. In a third Ames assay, pooled urine (0 to 2 mL/plate) from male Sprague-Dawley rats dosed for 15 consecutive days with 2000 mg/kg bw/day of di (2-ethylhexyl) terephthalate was tested with and without metabolic activation and also in the presence/absence of β-glucuronidase/aryl sulfatase. The latter treatment extended the sensitivity of the Ames assay to include hydrolyzed conjugates and their metabolites. Urine from treated rats showed no evidence of mutagenic activity under any test conditions.
In an in vitro chromosome aberration assay conducted by a method equivalent or similar to OECD Guideline 473, there was no evidence of cytotoxicity or an increase in the number of CHO cells with chromosomal aberrations in the presence or absence of metabolic activation at concentrations up to 1000 nL/mL (the highest concentration tested). In an in vitro CHO/HGPRT cell mutation assay conducted by a method equivalent or similar to OECD Guideline 476, there was no evidence of mutagenicity when cells were tested at concentrations up to 20 nL/mL in the presence and absence of activation. In this test, the highest two doses were cytotoxic with cell survival rates reduced to 69.2% and 72.9%, respectively, for the 20 and 10 nL/mL dose groups. For all studies, vehicle, negative and positive controls induced the appropriate responses. Di (2-ethylhexyl) terephthalate was not mutagenic/genotoxic under conditions used in these assays.
In addition to the five in vitro studies discussed above, a specialized in vivo study examining gene expression following in utero exposure to di (2-ethylhexyl) terephthalate was available for review. Pregnant Sprague-Dawley rats were treated by gavage daily from Gestational Days (GD) 12 through 19 with either corn oil (vehicle) or 500 mg/kg bw/day of di (2-ethylhexyl) terephthalate. At sacrifice on GD 19, fetal testes were harvested and global changes in gene expression were determined. There were no significant changes in gene expression under conditions of this assay for animals treated with di (2-ethylhexyl) terephthalate. By comparison, animals that were treated with di-(ethylhexyl) phthalate (DEHP) exhibited significant alterations in gene pathways responsible for cholesterol transport and steroidogenesis, intracellular lipid and cholesterol homeostasis, insulin signaling, transcriptional regulation, oxidative stress, alpha inhibin (essential for normal Sertoli cell development), and genes involved with communication between Sertoli cells and gonocytes.
Endpoint Conclusion: No adverse effect observed (negative)
Based on negative results in five in vitro studies conducted in bacterial or mammalian cells as well as an in vivo study which failed to detect adverse effects on gene expression in fetal testes when dams where exposed to di (2-ethylhexyl) terephthalate during gestation, the total weight-of-the-evidence indicates that di (2-ethylhexyl) terephthalate is not expected to induce heritable mutations in the germ cells of humans. Di (2-ethylhexyl) terephthalate was not previously classified under Directive 67/548/EEC, i.e., Annex I of the Dangerous Substances Directive for mutagenicity/genotoxicity. Based on a weight-of-the-evidence assessment, di (2-ethylhexyl) terephthalate would not be classified “Mutagenicity/genotoxicity” according to the UN Globally Harmonized System of Classification and Labeling (GHS) or the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) no. 1272/2008.
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