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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non Genotoxic based on in vitro studies

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliable without restriction; study was conducted by methods similar to OECD Guideline 471.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Guideline states E. coli WP2 uvrA, E. coli WP2 uvrA (pKM101), or S. typhimurium TA 102 should be one of the strains used in standard testing. In this study, S. typhimurium 1538 was used as 5th tester strain. This does not impact the quality of the study.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
His (-)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The tester strains were obtained from Dr. Bruce Ames (University of California, Berkeley, CA)
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
The tester strain was obtained from Dr. Bruce Ames (University of California, Berkeley, CA)
Metabolic activation:
with and without
Metabolic activation system:
Induced Rat Liver S9 (no further details regarding preparation of the metabolic activation system were provided in the publication).
Test concentrations with justification for top dose:
Screening Study:
10 doses from 0.32 to 10000 µg/plate

Mutagenicity Study:
1.0, 10, 100, 1000, 10000 µg/plate
Vehicle / solvent:
ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-n'-nitro-n-nitrosoguanidine
Remarks:
N-methyl-n'-nitro-n-nitrosoguanidine was used with tester strains TA 100 and TA 1535 at dose concentrations of 0.75 and 2.0 µg/plate, respectively, without metabolic activation.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9-Aminoacridine was used with tester strain TA 1537 at a dose concentration of 75 µg/plate without metabolic activation.
Positive controls:
yes
Positive control substance:
other: Picrolonic acid
Remarks:
Picrolonic acid was used with tester strain TA 1538 at a dose concentration of 200 µg/plate without metabolic activation.
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
ICR-191 was used with tester strain TA 98 at a dose concentration of 3.0 µg/plate without metabolic activation.
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2-Aminoanthracene was used with tester strains TA 1535, TA 1537, and TA 1538 at 2.5 µg/plate and TA 98 and TA 100 at 0.6 µg/plate with metabolic activation.
Details on test system and experimental conditions:
Screening Assay:
The screening study used tester strain TA 100 and half-log dose intervals of the test substance from 0.32 µg/plate to 10,000 µg/plate. Tester strain TA 100 was used because of its high spontaneous reversion rate. Spontaneous revertant numbers were counted and plotted against the dose of the test substance, producing a survival curve for the his+ genotype. The three highest doses designated for the mutagenicity assay were those which produced 10, 50, and 90% survival and the lowest two doses were 1/10 and 1/100 of the amount giving 90% survival in the toxicity assay. In this case, where toxicity was absent, doses from 10,000 µg/plate to 1.0 µg/plate were tested.

Mutagenicity Assay:
The mutagenicity assay was performed by mixing approximately 10^8 cells from an overnight tester strain growth culture with a known amount of the test substance, the induced rat liver S9 mixture (when required), and top agar containing a minimal amount of histidine. The resulting mixture was poured onto the surface of a sterile Petri dish containing 25 mL of solidified bottom agar and incubated for 48 hours at 37 °C. Revertant colonies were counted using a Biotran automated colony counter. Vehicle control plates contained no added test chemical and positive control plates containing appropriate amounts of chemicals known to be active were also performed with each tester strain. All platings were done in triplicate.
Evaluation criteria:
A response was considered to be positive if there was a dose-dependent increase in revertants per plate resulting in
(1) at least a doubling of the background reversion rate for strains TA 98 or TA 100, or
(2) at least a tripling of the background reversion rates for strains TA 1535, TA 1537, or TA 1538.
Statistics:
Statistical analyses were not needed due to the absence of an increase in the number of revertant colonies at any dose level beyond that seen with the positive control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: No cytotoxicity was observed at concentrations up to 10,000 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: No cytotoxicity was observed at concentrations up to 10,000 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The results of the experiment conducted on the test substance with and without metabolic activity were all negative at dose concentrations up to 10,000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It is concluded that, under the conditions of this test, di (2-ethylhexyl) terephthalate showed no evidence of mutagenic activity in this bacterial system with and without S9-induced metabolic activation at dose concentrations up to 10,000 µg/plate.

Based on the absence of genotoxic or mutagenic effects in this study with and without metabolic activation, di (2-ethylhexyl) terephthalate is not classified for “Germ Cell Mutagenicity” according to GHS.
Executive summary:

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. 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliable without restriction; the study was conducted according to GLPs and methods similar to those outlined in OECD Guideline 473.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
No guideline deviations; one revision was made to the final report, but it did not impact the quality or integrity of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cells used in the study were Chinese hamster ovary cells (CHO-WBI). The cells used in this study were obtained from Dr. S. Wolff, University of California, San Francisco, California, USA. and were cloned in Dr. A. Bloom’s Laboratory, Columbia University, New York, NY, USA.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
Preliminary range-finding study:
0, 100, 333, 1000 nL/mL

Chromosome Aberration study:
0, 700, 800, 900, and 1000 nL/mL
Untreated negative controls:
yes
Remarks:
McCoys 5a culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol (20 µL/mL)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Mitomycin C (0.5 and1.0 µg/mL) was used as the positive control chemical for treatment without induced rat liver S9 activation.
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide (50 µL/mL) was used as the positive control chemical for treatment with induced rat liver S9 activation.
Details on test system and experimental conditions:
For all evaluations, cultures were prepared by seeding 1.5 x 10^6 cells in 75 cm^2 flasks for approximately 24 hours prior to treatment in McCoy's 5a medium supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics. Negative controls, solvent controls, and positive controls were included in these assays. The negative controls contained only cells and the culture medium. The solvent controls contained the solvent ethanol, at a final concentration of 20 µL/mL. The positive controls for both the trials with and without metabolic activation were dissolved in water at the concentrations specified above.

For the Range-finding assay without metabolic activation, cultures were prepared by seeding 0.8x10^6 cells in 25 cm^2 flasks in 5 mL medium. The resulting 5mL cultures were exposed to the test chemical in a series of doses over four orders of magnitude. The top dose was limited by solubility and the lower doses formed a half-log series. One day after culture initiation, cells were treated with the test chemical for 2 hours. Then 5-bromo-2’-deoxyuridine (BrdU: 10µM) was added to cultures and incubation was continued in the dark for 23 hours. Cell monolayers were washed with phosphate-buffered saline and then medium with BrdU and colcemid (0.1 µg/mL) was added. Cultures were incubated for an additional 2.5 hr. Metaphase cells were collected by mitotic shake-off, cells were swollen with 0.075M KCl hypotonic solution, washed three times in a methanol:acetic acid (3:1) fixative, dropped on slides, and air dried.

For the Range-finding assay with metabolic activation, cells were incubated for 2 hr in the presence of test material and S9 reaction mix in growth medium without calf serum. After 2 hr, cells were washed at least twice with buffered saline, and normal growth medium containing 10% fetal calf serum (FCS) and 10 µM BrdU was added. Cultures were incubated for 23 additional hr, then colcemid was added. After 2.5 hr, metaphase cells were collected and fixed as described above.

For the Chromosome aberration assay without metabolic activation, cultures were treated with the test substance for 7.6 hours. Cultures were then washed with saline and fresh culture medium was added with colcemid (0.1 µg/mL). After 2.5 hr, metaphase cells were collected by mitotic shakeoff , cells were swollen with hypotonic KCl solution, and washed three times in fixative; slides were prepared as described for the range-finding test.

Estimation of cell cycle delay: Slides were stained for 10 minutes with Hoechst 33258 in phosphate buffer, mounted in the same buffer and exposed at 55-60 °C to “black-light” for about 3-10 minutes. Slides were then stained with Giemsa for 5-20 minutes and air-dried.

Aberration assays: Duplicate 10 mL cultures were used for each dose level.
For the Chromosome aberration assay in the presence of metabolic activation, on the day after culture initiation, cultures were treated with the test substance and the S9 reaction mix in growth medium without fetal calf serum for 2 hours. After 2 hours, cells were washed twice with buffered saline, and growth medium containing 10% FCS was added. Cultures were incubated for 8.1 additional hours, with colcemid present for the last 2.5 hours. Metaphase cells were collected and fixed as described above.


Staining and scoring of slides: Slides were stained with 5% Giemsa for scoring of chromosome aberrations. Slides were coded and scored “blind” to control for bias. Two hundred cells were scored per concentration level (100 from each replicate) of test material in all of the study phases. For the positive controls, 25 to 50 cells were scored from one dose level.

-Study Dates:
Experimental Starting Date: August 6, 1984
Experimental Completion Date: September 24, 1984
Evaluation criteria:
A response was considered to be positive if a dose-dependent increase in aberration frequency was observed for the treated cultures. Cells were evaluated for 14 common aberrations.
Statistics:
Statistical analysis employed the Student t-test to compare the aberration frequency in treated cells with the solvent and negative controls. Statistical significance was ascribed at p<0.05.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: No cytotoxicity was observed at concentrations up to 1000 nL/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the screening assay, concentrations of di (2-ethylhexyl) terephthalate from 100 nL/mL to 1000 nL/mL caused slight clouding of the culture medium but no reduction in mitotic activity or in monolayer confluency with and without activation. When di (2-ethylhexyl) terephthalate was tested for cell cycle delay at concentrations of 100, 333 or 1000 nL/mL, no cell cycle delay was observed and a 10-hr harvest time was chosen for the aberrations assay with a dose range of 700-1000 nL/mL.

In the aberrations assay in the cultures treated with di (2-ethylhexyl) terephthalate, no statistically significant increases in aberrations were observed at any dose level and there was no toxicity observed at any dose level. Di (2-ethylhexyl) terephthalate at all dose concentrations tested resulted in a slight clouding of the culture medium but this clouding did not interfere with the ability to observe chromosome aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Di (2-ethylhexyl) terephthalate showed no evidence of clastogenic activity when tested in an in vitro cytogenetic test with Chinese hamster ovary cells (CHO-WBI) at concentrations up to 1000 nL/mL with and without metabolic activity.

Based on an absence of genotoxic/mutagenic effects in this study, di (2-ethylhexyl) terephthalate is not classified for “Germ Cell Mutagenicity” according to GHS.
Executive summary:

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.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliable without restriction; the study was conducted in compliance with the GLPs and was conducted similar to OECD Guideline 476.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Study was conducted using a procedure similar to the one described in the OECD Guideline 476, "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests" (1984); and the study was performed using the methods described by O'Neill et. al. (Mutat. Res. 45:91, 1977)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The study report states that the cells were Chinese hamster ovary cells (CHO-K1-BH4), obtained from A. Hsie, Oak Ridge National Laboratory, Oak Ridge, TN.
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver S9
Test concentrations with justification for top dose:
Preliminary cytotoxicity assay:
0, 0.001, 0.002, 0.005, 0.01, 0.02, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 nL/mL

Mutation Assay:
0, 1.25, 2.5, 5, 10, 20 nL/ml
Vehicle / solvent:
Ham's F12 culture medium supplemented with L-glutamine and heat-inactivated dialyzed fetal bovine serum (5% by volume)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% Dimethyl sulfoxide used with and without induced rat liver S9 activation.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethanesulfonate (0.25 mg/mL) was used as the positive control chemical for treatment without induced rat liver S9 activation.
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
Dimethylnitrosamine (0.25 mg/mL) was used as the positive control chemical for treatment with induced rat liver S9 activation.
Details on test system and experimental conditions:
Doses used in the mutagenicity assay were selected following a cytotoxicity assay where 10-15 doses of the test material were tested for clonal cytotoxicity in Ham’s F12 culture medium. Five concentrations were then selected covering the range from 0% to 90% reduction of colony forming ability. For the mutagenicity assay, 5 x 10^5 cells were seeded in 25 cm2 flasks and incubated for approximately 24 hours. After incubation, media containing the test substance or a positive or solvent control was added 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 approximately 100 cells per flask. These flasks were incubated for 10 days to determine cell survival from the treatments. Approximately 10^6 cells were seeded in 75 cm2 flasks and subcultured for 9 days to allow for the expression of induced mutations. At the end of the expression period, the cultures were reseeded at a density of 2 x 10^5 cells per flask in F12 medium containing 10 µM 6-thioguanine as the selective agent. After 7 days of incubation, the colonies were stained and counted and the results were recorded.
Evaluation criteria:
The mutant frequency at each treatment condition was calculated by dividing the total number of mutant colonies observed by the product: [number of dishes] x [2 x 10^5 cells per dish] x [absolute cloning efficiency]. Acceptability criteria for this assay included that 1) a minimum of 10^6 cells are available for evaluation at the end of the test period; 2) at least three dose levels of the test compound are available for evaluation at the end of the test; 3) the mean number of mutants in the positive control groups are significantly different from the solvent control groups; and 4) the mutant frequency in the solvent control group does not exceed 20 mutants per 10^6 cells.
Statistics:
Mutant frequency data were evaluated using the statistical tables provided by Kastenbaum and Bowman [1970].
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the clonal cytotoxicity assay, fifteen doses of di (2-ethylhexyl) terephthalate ranging from 0.001 to 20 nL/mL were used. Cell survival from 88.6 to 106.7% of the control was observed, indicating no reduction in cloning efficiency at doses of up to 20 nL/mL. On the basis of clonal cytotoxicity, dose levels of 0, 1.25, 2.5, 5, 10, 20 nL/mL were used for the mutagenicity assay. During the treatment phase of the mutagenicity assay, the relative cell survival rates (in the absence of metabolic activation) were reduced to 69.2% and 72.9% for the 20 and 10 nL/mL dose groups, respectively, indicative of a weak toxic response. The positive control substances used with and without metabolic activation also reduced the relative cell survival rates to 34.8% and 40.4%, respectively. However, cell survival rates for all other treatment groups with and without metabolic activation were comparable during the study.

In the cultures treated with di (2-ethylhexyl) terephthalate, there were no statistically significant increases in mutation frequencies when compared with the solvent controls both with and without metabolic activation. In contrast, the mutation frequency was significantly elevated with cultures containing the positive control materials under conditions both with and without rat liver S-9 activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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.

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

Di (2-ethylhexyl) terephthalate was not mutagenic in the CHO/HGPRT Forward Mutation Assay when tested at concentrations up to 20 nL/mL in the presence or absence of metabolic activation using rat liver S-9. In a preliminary cytotoxicity assay, there was no reduction in cloning efficiency at doses levels up to 20 nL/mL. In the actual mutagenicity assay, relative cell survival rates were reduced to 69.2% and 72.9% for the 20 and 10 nL/mL doses groups, respectively, indicating a weak toxic response.

Based on an absence of genotoxic/mutagenic effects even at cytotoxic dose levels, di (2-ethylhexyl) terephthalate is not classified for “Germ Cell Mutagenicity” according to GHS.

Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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)

Justification for classification or non-classification

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.